Team:Freiburg Bioware/NoteBook/Labjournal/August

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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 145 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 146 - 155 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3">October part 3 (labday 156 - 166 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture </a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
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Latest revision as of 21:36, 27 October 2010

=> Back to Notebook overview

Contents

76.Labortag 01.08.2010

Picking clones of pSB1C3_CMV

Investigator: Bea

Comments: Trafo was performed friday, and trafo plate were incubated over night. Clones need to be picked in order to perform Mini-Prep

  • Bacterial strain used: XL1-B
  • Two clones were picked of trafo plate
  • Inoculating of 10 mL DYT medium containing 10µL chloramphenicol
  • Put in 37°C room on rotary shaker


77.Labortag 02.08.2010

Sequenzing of pSB1C3_RFC25_longlinker, pSB1C3_RFC25_SEG, pSB1C3_RFC25_GSAT

Investigator: Jessica

Comments: Linkers (we got from Gerrit) we cloned in the RFC25-standard (pSB1C3_RFC25). Result looks good, linkers are in the vector pSb1C3_RFC25

  • pSB1C3_RFC25_longlinker P105 und P106
  • pSB1C3_RFC25_SEG P107 und P108
  • pSB1C3_RFC25_GSAT P109 und P110

Freiburg10 Sequence alignment longlinker.jpg
Freiburg10 Sequence alignment SEG.jpg
Freiburg10 Sequence alignment GSAT.jpg



Sequencing of pGA14_Prefix-leftITR

Investigator: Hanna

Eventually GATC partially managed to sequence pGA14_Prefix-left ITR clone 1 and 2.
The sequencing results were better than before but in the ITR region still not evaluable due to the strong secondary structures.

It seemed that, despite of successful insertion of the RFC10-Prefix, something went wrong because there were 2 bases missing in the NotI restriction site. But by having a closer look, it became obvious that the Geneious misinterpreted the chromatogram and overlaid the peaks of the missing bases.

LeftITR+Prefix.jpg



For the first time sequencing of parts of the 3' end worked: The remaining NotI restriction site, which was already eliminated this week and also the "backbone-PstI" can be seen:

LeftITR+Prefix 2.jpg



Sequencing of very GC-rich regions :) :) :)

LeftITR+Prefix 3.jpg



Sequencing of clone 4 delivered that the ITR and the RFC10-Prefix were not inserted in the vector. The RFC25 standard of the multiple cloning site is still in the plasmid:

LeftITR+Prefix clone4.jpg



Therefore clone 4 was dicarded from the plasmid- and glycerol stock.

Repetition of test digestion pSB1C3_CFP SDM SspI and PvuII

Investigator: Jessica

  • buffer used: 2; Restriction-enzymes used: Enzyme 1: SspI ; Enzyme 2: PvuII
  • Plasmids (all: ~ 700 ng/µL):
    • pSB1C3_CFP P51.1
    • pSB1C3_SDM_SspI P125
    • pSB1C3_SDM_PvuII P129
    • pSB1C3_SDM_PvuII P131



Components Mastermix P125/µL P51.1.1/µL P129/µL P131/µL P51.1.2/µL
DNA - 2,4 2,3 1,6 5,35,3
BSA (10x) 6 1 1 1 1 1
Buffer 2 (10x) 6 1 1 1 12 1
Enzyme 1 SspI (68) - 1 1 - - -
Enzyme 2 PvuII (50) - - - 1 1 1
H2O - 4,6 4,7 5,4 1,7 1,7
Total volume 12 10 10 10 10 10


  • Incubation: 65 minutes

Agarosegel
0.45 g Agarose, 50 ml TEB (0,5 %), 3 µL GELRED, at 115 Volt, running time: 45 minutes

  • Marker: GeneRuler ladder mix (6x)
Marker P125 P51.1 SspI - P129 P131 P51.1 PvuII
Lane 7 µL 10 µL 10 µL - 10 µL 10 µL 10 µL


Freiburg10 Gel test digestion SDM.jpg
























  • P125 isn't cut, therefore you see the bands for supercoiled, nicked and relaxed
  • P51.1 is cut once, therefore you can see a faster running band
  • P129 and P131 should be cut once because of the PvuII in the CFP but there is another band we can't identify
  • P51.1 should be cut two times but there is also another unidentified band
    • perhaps there is another PvuII in the vector we can't find (???)

Comments: SDM of SspI is ready! SDM of PvuII looks confusing because of the lower band. P 129 will be sequenced but this doesn't solve this problem. there has to be one more rs we don't know, perhaps. we will check it...anywise


Mini-Prep and Test digestion of pSB1C3_CMV

Investigator: Kerstin
Nanodrop

  • pSB1C3_CMV clone1: 157,6 ng/µl P145
  • pSB1C3_CMV clone2: 155,6 ng/µl P146



Test digestion 900ng DNA

components P145 /µl P146 /µl
DNA 5,75,8
BSA (10x) 1,5 1,5
Buffer 4 (10x) 0,5 0,5
Enzyme 1: XbaI 0,5 1,5
Enzyme 2: PstI 0,5 0,5
H2O 5,3 5,2
Total volume (e.g. 15,20,25,30 µl) 15 15


agarose gel: 1,5%, digestion: 90 minutes, 37°C

Freiburg 10 pSB1C3 CMV.jpg




P145 is sent for sequencing with Reverse Primer (VR2)

Comments:Results look good. CMV has the expected size (~ 662bp)


</p>

Sent for sequencing

Investigator: Jessica
Hanna

  • P145 left ITR
    • Primer: GATC_std_pQE-FP and GATC_std_m13-FP
  • P150 right ITR
    • Primer: GATC_std_pQE-FP and GATC_std_m13-FP


Anissa

  • P136 pSB1C3_hgh
    • Primer: O51_Reverse Primer (VR2)
  • P139 pAAV_BamHI
    • Primer: O36_Rep_1250_rev
  • P143 pAAV_RC_SalI
    • Primer: O53_Rep_1250_for
  • P141 pGL3_QTERT
    • Primer: GATC_std_pTal-FP
  • P133 pSB1C3_betaGlobin
    • Primer: O51_ReversePrimer (VR2)
  • P145 pSB1C3_CMV
    • Primer: O51_ReversePrimer (VR2)


Jessica

  • P129 pSB1C3_SDM_PvuII
    • Primer: GATC_std_pTeSp-2


Preparation of competent E.coli

Investigator: Jessica

  • 15ml DYT was prepared with 15µl tetracycline and inoculate with XL1B
  • 10ml DYT w/o antibiotics was prepared and inoculate with BL21 (just for glycerol stock, no competent cells)

both were incubate over night in 37°C room

Last Mini-Prep and test digestion of ITRs

Investigator: Hanna

Plasmid Mini-Prep

  • new vector name: pGA14_RFC10_leftITR and pGA14_RFC10_rightITR


Glycerol Stocks
pGA14_RFC10_leftITR

Clone 1 Clone 2 Clone 3
Bacteria strain XL1blue XL1blue XL1blue
Plasmidname pGA14_RFC10_leftITR pGA14_RFC10_leftITR pGA14_RFC10_leftITR
Date 2.8.10 2.8.10 2.8.10
given glycerol-stock no. B115 B116 B117
given plasmid no. P147 P148 P149


pGA14_RFC10_rightITR

Clone 1 Clone 2 Clone 3
Bacteria strain XL1blue XL1blue XL1blue
Plasmidname pGA14_RFC10_rightITR pGA14_RFC10_rightITR pGA14_RFC10_rightITR
Date 2.8.10 2.8.10 2.8.10
given glycerol-stock no. B112 B113 B114
given plasmid no. P150 P151 P152


Test digestion

  • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 XbaI ; Enzyme 2 SpeI
  • Plasmid: pGA14_RFC10_leftITR:
    • Given Plasmid-Number: P147; DNA concentration: 240.22 ng/µL ;
    • Given Plasmid-Number: P148; DNA concentration: 268.97 ng/µL ;
    • Given Plasmid-Number: P149; DNA concentration: 234.16 ng/µL ;


  • Plasmid: pGA14_RFC10_rightITR:
    • Given Plasmid-Number: P150; DNA concentration: 221.71 ng/µL ;
    • Given Plasmid-Number: P151; DNA concentration: 191.13 ng/µL ;
    • Given Plasmid-Number: P152; DNA concentration: 215.14 ng/µL ;



Components P147 Volume/µL P150 Volume/µL
DNA 4.2 4.5
BSA (10x) 1 1
Buffer no. 4 (10x) 1 1
Enzyme 1 XbaI 0.5 0.5
Enzyme 2 SpeI 0.5 0.5
H2O 2.8 2.5
Total volume P147 P150


  • Incubation: 1 h


Agarose-Gel:


0.83 g Agarose, 53 mL TBE (1.57%), 3 µL GELRED, at 115 Volt, running time: 45 minutes

Sample Sample/µl] Loading dye (5x)/µl Expected size 1 (Geneious) Expected size 2 (Geneious)
P147 10 µl 2 µl 157 bp 2902 bp
P150 10 µl 2 µl 156 bp 2903 bp


  • Marker: GeneRuler ladder mix
Marker /µL Sample P147 /µL Sample P150 /µL
Lane 6.5 12 12



Test digestion delivered positive results for both ITRs: The expected fragment sizes (156 resp. 157 bp) could be detected in the referring range between the 100 and 200 bp marker nucleotides:


LastITRPrep.jpg


Comment: In order to verify the results, P147 and P150 were sent for sequencing. Because sequencing never worked before, they will be sequenced under special conditions this time.

BioBrick production: mGMK in pSB1C3

Investigator: Stefan
Backbone taken from:

  • pSB1C3_CFP (P51.2): 151,1 ng/µl

Insert taken from:

  • pAAV_RFC25_mGMK (P100): 411,3 ng/µl


components pSB1C3_CFP(P51.2) /µlpAAV_RFC25_mGMK (P100) /µl
DNA 8 5
BSA (10x) 2 2
Buffer 4 (10x)2 2
XbaI 1 1
AgeI 1 1
H2O6 9
Total volume 20 20


  • 1% Agarose gel
  • 3 µl Gelred
  • 7 µl DNA-Ladder-Mix
  • 115 Volt, running time: 50 minutes

Afer 50 minutes a picture (see below) was taken. Because there was no clear pattern (two bands) for the pSB1C3 backbone, the band of the insert mGMK was cut out and the gel ran another 20 minutes. After that, there were still two bands. Both were cut out and it was continued with two possible backbones. They were labeled vector (lower) and vector (upper).

Freiburg10 BioBrick pSB1C3 mGMK.jpg

Sample/µl Expected size/bp
Vector pSB1C3 2082
Insert mGMK 603


  • weight of insert mGMK gel extract: 0,17 g
  • weight of vector (lower) pSB1C3 gel extract: 0,08 g
  • weight of vector (upper) pSB1C3 gel extract: 0,09 g

Nanodrop

  • insert mGMK : 4,13 ng/µl
  • vector (lower) pSB1C: 5,47 ng/µl
  • vector (upper) pSB1C: 0,60 ng/µl => for calculation of ligation approach calculated with 1 ng/µl


Ligation

  • vector (lower) : insert - 4,16 µl : 4,84 µl
  • vector (upper) : insert - 7,43 µl : 1,57 µl

Transformation Two approaches were prepared, one for each vector and put in 37 °C room overnight.

Sequencing results of SDM Rep 68 & 78

Investigator: Hanna and Volker
The sequencing results delivered that the PstI restriction sites of Rep 68 and Rep 78 were successfully deleted via side-directed mutageneis:

78.Labortag 03.08.2010

Results of sequencing pSB1C3_CFP_SDM_PvuII

Investigator: Jessica
SDM of PvuII:

PvuII.jpg



PvuII is succesfully deleted in the vector pSB1C3_RFC25_CFP

Repetition of the quickchange site-directed mutagenesis of pAAV_RC_1.1_SalI

Investigator: Kerstin, Anissa

Comments: Sequenzing revealed no mutagenesis of SalI in pAAV_RC_1.1_SalI, so SDM now will be repeated.


PCR-reaction:

Volume / µl ingredientsrecommended /µl
2,5 10X Pfu Ultra II buffer 2,5
4,18 template (~10 ng) 4,18 of 1:100 dilution
0,58forward primer: O68 62,5 ng
0,58 reverse primer: O69 62,5 ng
0,5dNTP 250 µM each dNTP
16,16H2O
0,5PfuUltra II fusion (1.25)


PCR program:

Roundstemperature/ °CTime
1 95 2 minutes
20 9530 seconds
20 80,4 1 minute
2068 7,5 minutes


Plasmid was transformed into BL21 cells. Clones have to be picked tomorrow.

Comments:NO CLONES, trafo didn't work, because annealing temperature was totally to high! (80,4 °C instead of 55°C) annealing temperature should always be 55°C (in case of troubleshooting temperature can be increased up to MAX. 68°C !)


PCR for Biobrickproduction of Rep 40, 52, 68, 78 and APP

Investigators: Volker, Anna


Aim of the experiment: We wanted to produce biobricks from the expression plasmids Rep40ex, Rep52 ex, the Rep 68 and 78 in which the PstI at position 310 was silenced and the AAP. This was performed by a PCR with primes that annealed to the ends of the later biobrick but also contained restriction sites that can be cut afterwards and cloned into pSB1C3.

  • Plasmids used as template:

Rep_68_ex (p119): c = 470,6 ng/µl
Rep_78_(p122): c = 201,08 ng/µl
Rep_40_(p22.2): c = 673,1 ng/µl
Rep_52_(p23.2): c = 532,22 ng/µl
pAAV-RC containing AAP ORF (p50): c = 378,5 ng/µl


  • Primer used:

For Rep_40_ex: Praefix_40_52_ex & Suffix_40_68_ex
For Rep_52_ex: Praefix_40_52_ex & Suffix_52_78_ex
For Rep_68_ex: Praefix_68_78_ex & Suffix_40_68_ex
For Rep_78_ex: Praefix_68_78_ex & Suffix_52_78_ex
For AAP_ex: Praefix_AAP_ex & Suffix_AAP_ex


  • PCR:

(was performed following the standard protocol)

Ingredients Volume / µl Rep68Rep78 Rep40Rep52AAP
5X Phusion HF buffer 10
10 mM dNTP mix1
forward primer: 2,5
reverse primer: 2,5
DNA Template***4,2 µl 1 µl 2,9 µl3,8 µl5,3 µl
Phusion Polymerase0,5
H2O*** 28,3 µl 31,5 µl 29,6 µl 28,7 µl27,2 µl
Total volume50


PCR program:

PCR Programtemperature/ °CTime Rep40Rep50Rep68Rep78AAP
198 1min
298 15s
8x*** 25s63°C62°C63°C62°C64°C
3 72***15s 18s24s 27s 10s
4 98 15s
17x***25s68°C66°C68°C64°C68°C
572***15s18s24s27s10s
6x725min
Hold4


Five µl of PCR-product were used for an analytical gel to see if the PCR worked.

Comment: The result is that the PCR worked for the longer Rep proteins (68&78) with which a Quick-change was performed to remove the PstI(310) but not with the constructs for the smaller Rep variants (40&52) that were recieved from PD Kleinschmidt. As the longer Rep variants the AAP PCR Reaction also resulted in a PCR-product of the expected size. Unfortunately there were a secondary band of ~90bp for Rep_68_ex. For this reason we performed a praeparative gel and decided to cut the bands of all PCR products.


The bands marked in the gel picture were cut and a gel extraction was performed.

Gel extraction


Gel measurement:

Sample Weight Volume Concentration
Rep_68_ex 0,1 g 20 µl 17,6 ng/µl
Rep_78_ex 0,15g 20 µl 80,6 ng/µl
AAP_ex 0,19 g 20µl 77,78 ng/µl

Continuation of preparation of competent E.coli

Investigator: Jessica
preparation of competent XL1blue was finished according to the standard protocol

  • aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer

Sequencing of pSB1C3

Investigator: Jessica

Comments: We sent for sequencing pSB1C3_RFC25_longlinker (P105) to check the strange result of the test digestion of pSB1C3_SDM_PvuII(01.08.10.
pSB1C3_SDM_PvuII is too long for sequencing wherefore we sent pSB1C3_RFC25_longlinker because the longlinker just have 54kb (bp, nicht kb (Volker) (necessary is just the backbone we don't have.

  • Primer:
    • RESgen-241698
    • Reverse primer (VR2)
    • o_F1-fw (from Gerrit)



Design of Primer for p5 Promoter WT and TATA-less BioBrick production

Investigator: Hanna

Freiburg10 p5 Primer.JPG


Freiburg10 p5Primer Aim.JPG

79.Labortag 04.08.2010

Cloning of right ITR and left ITR into pSB1C3_RFC25_CFP

Investigator: Patrick
Intention: Get biobricks ready.
pGA14_lITR_RFC10 (P147), pGA14_rightITR (P150) and pSB1C3_RFC25_CFP (P51.1) were digested with EcoRI and PstI (Buffer 4) according to the standard protocol. Digestion Time: 80 minutes.
GelRun: 1% Agarose Gel. Expected results (from left to right):

  • pSB1C3_RFC25_CFP: about 2100 bp and 800 bp.
  • pGA14_leftITR_RFC10: the size of the insert should be 135 bp.
  • pGA14_rightITR_RFC10: the size of the insert should be 138bp.
20100704 patrick bearbeitet.JPG


The mutual vector (now without CFP) and inserts (left ITR and right ITR) were cut out. The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

  • pSB1C3_RFC25_CFP: 2,9 ng/µl
  • left ITR: 1,4 ng/µl
  • right ITR: 2,0 ng/µl


The Quick Ligation was not performed according to the standard protocol:

  • left ITR + pSB1C3: 5µl Buffer, 1 µl Quick-Ligase, 2,5 µl left ITR, 1,5 µl pSB1C3_RFC25.
  • right ITR + pSB1C3: 5µl Buffer, 1 µl Quick-Ligase, 2,5 µl right ITR, 1,5 µl pSB1C3_RFC25.


Transformation: performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol. The clones will be picked tomorrow.

Cloning of SDM SspI and SDM PvuII

Investigator: Jessica

  • Vector: name: pSB1C3_SDM_SspI P125
  • Insert: name: pSB1C3_SDM_PvuII P129
  • new vector name: pSB1c3_SDM_SspI/PvuII P157
  • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 141) BstI ; Enzyme 2 (no.Lab: 144) BpmI
  • DNA concentration (vector): 321,9 ng/µl ; DNA concentration (insert): 308,4 µg/µl


components volume of pSB1C3_SDM_SspI /µl volume of pSB1C3_SDm_PvuII /µl
DNA 4,66 4,87
BSA (10x) 2 2
Buffer 4 (10x)2 2
Enzyme BstI (no.Lab:141)1 1
Enzyme BpmI (no.Lab:144)1 1
H2O9,34 9,13
Total volume (e.g. 15,20,25,30 µl) 2020


0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45


Loading plan for agarose gel:
Marker used: GeneRuler ladder mix (Fermentas)

Marker Sample 125, 20µl Sample 129, 20µl
Lane 1 3 5


Results: digesttemperature of BstI is 55°C, plasmid was just digested at 37°C... :( (

Digestion of SDM SspI and SDM PvuII.jpg

2nd Repetition of Quickchange site-directed mutagenesis of pAAV_RC_1.1_SalI

Investigator: Kerstin, Anissa

Comments: Last trafo didn't work. Annealing temperature was to high (80,4°C instead of 55°C)


Two approaches were made (short and long PCR:

PCR-reaction:

Volume / µl ingredientsrecommended /µl
2,5 10X Pfu Ultra II buffer 2,5
1,83 template (~10 ng): p139 (c: 546,97 ng/µl) 1,83µl of 1:100 dilution
0,58forward primer: O68 62,5 ng
0,58 reverse primer: O69 62,5 ng
0,5dNTP 250 µM each dNTP
18,51H2O
0,5PfuUltra II fusion (1.25)


PCR program (long):

Cyclestemperature/ °CTime
1 95 2 minutes
20 9530 seconds
20 55 1 minute
2068 7,5 minutes


PCR program (short):

Cyclestemperature/ °CTime
1 95 2 minutes
20 9530 seconds
20 55 1 minute
2068 4 minutes

Plasmids were transformed into BL21 cells. Clones have to be picked tomorrow.

Mini-Prep of pSB1C3_mGMK

Investigator: Chris W., Bea


Vector name: pSB1C3_mGMK upper band 1.1 and pSB1C3_mGMK upper band 1.2 and pSB1C3_mGMK lower band 2.1 and pSB1C3_mGMK lower band 2.2

Mini-Prep following the standart Protokoll

  • P153 = pSB1C3_mGMK upper band 1.1 = 248,3 ng/µl
  • P154 = pSB1C3_mGMK upper band 1.2 = 251,6 ng/µl
  • P155 = pSB1C3_mGMK lower band 1.1 = 263 ng/µl
  • P156 = pSB1C3_mGMK lower band 1.2 = 235,2 ng/µl


    Test digestion:
    • Perfomed with 15 µL of total volume with all four clones
    •   Mastermix/µL P153/µL P154/µL P155/µL P156/µL
      DNA - 3,5 3,5 3,5 3,5
      BSA (10x) 7,5 4 4 4 4
      Buffer 4 (10x) 7,5
      Enzyme XbaI 2,5
      Enzyme AgeI 2,5
      H2O - 7,5 7,5 7,5 7,5
      Total volume 15 15 15 15 15


    • All four samples were loaded on a 1% agarose gel
    • P153 = pSB1C3_mGMK upper band 1.1
    • P154 = pSB1C3_mGMK upper band 1.2
    • P155 = pSB1C3_mGMK lower band 1.1
    • P156 = pSB1C3_mGMK lower band 1.2


    Freiburg10 04 08 2010 Test digestion of pSB1C3 mGMK.jpg




    Sequencing:

    • Plasmid used: P156: pSB1C3_mGMK lower band 1.2
    • Primer used: VR-2
    • Tube name: Bea_1

    cell culture

    Investigator: Adrian
    Plan for the next virus production procedures
    The Motivation: investigation of the influence of different GOI amounts.
    The Plan: keep rep/cap and pHelper amount stable (3,3 µg each => 6,6 µg), differ the GOI (YFP) amount in each stock.
    Viral Stocks:

    • 3,3 µg YFP + 3,3 µg pHelper + 3,3 rep/cap
    • 10 µg YFP + 3,3 µg pHelper + 3,3 rep/cap
    • 20 µg YFP + 3,3 µg pHelper + 3,3 rep/cap


    • 3,3 µg pHelper + 3,3 rep/cap (without GOI !!!)


    • one GMK_TK clone (with the confirmed sequence) + 3,3 µg pHelper + 3,3 rep/cap


    Transduction plan

  • five 6-well-plates wille be transduced
    A 150µl AAV stock 1 300µl AAV stock 1 control no Virus
    B 150µl AAV stock 2 300µl AAV stock 2 150µl AAV without GOI

    Repetition of cloning of SDM SspI and SDM PvuII

    Investigator: Jessica

    • Vector: name: pSB1C3_SDM_SspI P125
    • Insert: name: pSB1C3_SDM_PvuII P129
    • new vector name: pSB1C3_RFC25_SDM_SspI/PvuII P157
    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 141) BtsI ; Enzyme 2 (no.Lab: 144) BpmI
    • DNA concentration (vector): 321,9 ng/µl ; DNA concentration (insert): 308,4 µg/µl


    components volume of pSB1C3_SDM_SspI /µl volume of pSB1C3_SDm_PvuII /µl
    DNA 4,66 4,87
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme ClaI (no.Lab:152)1 1
    Enzyme BpmI (no.Lab:144)1 1
    H2O9,34 9,13
    Total volume (e.g. 15,20,25,30 µl) 2020

    incubation time: 1,5h
    0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED (gel was shared with Anna) , at Volt, running time:


    Loading plan for agarose gel:
    Marker used: GeneRuler ladder mix (Fermentas)

    Marker Sample 125, 20µl Sample 129, 20µl
    Lane 1 3 5

    This appproach also didn't work, same result. the guess is that BpmI doesn't work anymore. will be repeated by Chris W. on 05.08.10. the result will show that the guess is right and BpmI from labstock is changed out with a new one.

    Test transformation of XL1 competent cells

    Investigator: Kira

    Test transformation was performed in order to test the efficiency of produced chemical competent XL1 blue cells.

    2 plates were prepared: one with 50 pg and the second with 100 pg pUC 18 plasmid (50 pg/㎕) 50 pg plate: 1 ㎕ pUC + 50 ㎕ XL1B cells 100 pg plate: 2 ㎕ pUC + 50 ㎕ XL1B cells

    Transformation was performed according to the standard protocol and the plates were incubated at 37 C.


    Sequencing results of ITRs

    Investigator: Hanna
    8 sequencing files were analyzed "per hand" base by base. Alignments (will be inserted soon) delivered that the right ITR is 100% OK and was successfully converted into the RFC10 BioBrick standard.

    Freiburg10 Sequencing RFC10RightITR 2.jpg

    The sequencing and alignments of the left ITR delivered that a 15 bp fragment in the middle of the sequence is lacking. Therefore further test digestions have to be performed. Nevertheless also this ITR was successfully converted into the RFC10 BioBrick standard. Secondary structure analysis showed that the stemloop-structure will be nevertheless forming. We will try to test whether the referring fragment is also missing in the pAAV_MCS. If it's also lacking there we will continue with this ITR and test whether it functions.

    Freiburg10 Sequencing RFC10LeftITR 2.jpg



    Cloning of Rep68, Rep78 and AAP into pSB1C3

    Investigator: Anna

    Comments: The PCR of Rep 40/52 didn't work (see 03.08), whereas the PCR of Rep 68/78 and AAP was succesful. Ligation was done with two different samples of pSB1C3 (see agarose gel). Samples from digestion and from ligation are stored in the 4°C freezer.


    • Digestion of PCR products and vector:


    components PCR product /µl vector /µl
    DNA 19 10
    BSA (10x) 3 3
    Buffer 4 (10x)3 3
    Enzyme XbaI 1 1
    Enzyme SpeI 1 1
    H2O3 12
    Total volume (e.g. 15,20,25,30 µl) 3030


    Comments: Digestion was done with XbaI and SpeI, it has to be checked if the inserts are cloned into the vector in the right orientation.


    • Purification of Rep68, 78 and AAP:

    For the purification 95 µl of buffer PBI was used.

    c(Rep68)= 17,6 ng/µl
    c(Rep78)= 80,60 ng/µl
    c(AAP)= 77,78 ng/µl


    • Gelextraction of pSB1C3_RFC25_CFP:

    0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:55
    5µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)



    Digestion of pSB1C3.jpg



    c(pSB1C3)= 2,87 ng/µl
    c(pSB1C3_2)= 9,46 ng/µl


    • Quickligation of PCR products and vector:

    For the Ligation 10µl buffer (2x) and 1µl Quickligase were used.

    Sample-no.vector /µl insert /µl
    pSB1C3 + Rep68 1.1 6,51 2,4
    pSB1C3_2 + Rep68 1.2 3,9 5,02
    pSB1C32 + Rep78 2.1 8,2 0,8
    pSB1C3_2 + Rep78 2.2 6,82 2,18
    pSB1C3 + AAP 3.1 8,71 0,92
    pSB1C3_2 + AAP 3.2 8,1 0,9
    Total volume (e.g. 15,20,25,30 µl) 9


    • Transformation:

    The transformation was done following the standard protocol using B21 cells.

    80.Labortag 05.08.2010

    New LB Agar was prepared.

    Investigator: Patrick

    Sequenc analysis of BioBricks: CMV, hGH and beta globin

    Investigator: Bea

    Comments: Sequence analysis of three BioBricks which were cloned into the iGEM standard plasmid pSB1C3. Cloning of this three plasmids were performed at:


    Sequencing results of pSB1C3_CMV:


    • Plasmid sent for sequencing:
    • Primer used: VR-2
    • Results: Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.But: there is a mutation in the termintor (sequence picture do not show this mutation)


    <img src="Freiburg10_Sequence_analysis_of_PSB1C3_CMV.jpg" />

    Sequencing results of pSB1C3_betaglobin:


    • Plasmid sent for sequencing:
    • Primer used: VR-2
    • Results: Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.

    Freiburg10_Sequence_analysis_of_PSB1C3_betaglobin.jpg

    Sequencing results of pSB1C3_hGH:


    • Plasmid sent for sequencing:
    • Primer used: VR-2
    • Results: Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.

    Freiburg10_Sequence_analysis_of_PSB1C3_hGH.jpg

    Cellculture

    Investigator: Adrian


    The Motivation: Transfection
    The Action: HEK cells were split into four T75 flasks
    The Plan: The cells should be ready at saturday for seeding and monday for transfection


    We recived new HT1080 cells, they'll be split tomorrow (thx to Sven)

    We recived new tumor cells which overexpress EGFR (thx to barbara)

    Picking clones of pSB1C3_AAP_2, pSB1C3_AAP, pSB1C3_Rep78_2, pSB1C3_Rep78, pSB1C3_Rep68_2 and pSB1C3_Rep68

    Investigator: Anissa
    Of each construct two clones were picked. plates are still stored in the cold-room, tomorrow mini-prep will be done.

    PCR and ligation for biobrick-production of pSB1C3_hTERT


    Investigator: Anissa,Kerstin


    Comments: PCR was performed one time without DMSO and one time with DMSO. Only the approach with DMSO showed bands in the analytic gel after PCR. That's why only this approach will be noted.



    • PCR:

    (was performed following the standard protocol)

    Ingredients Volume / µl
    5X Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: O111 (phTERT_prefix_for_RFC10) 2,5
    reverse primer: O112 (phTERT_suffix_rev_RFC10) 2,5
    DNA Template0,35 µl
    DMSO (2%) 1
    Phusion Polymerase0,5
    H2O32,15
    Total volume50


    PCR program:

    PCR Programtemperature/ °C
    198
    298
    8x58
    3 72
    4 98
    17x70
    572
    6x72
    Hold4




    Gel extraction


    Gel measurement:

    Sample Weight Volume Concentration
    hTERT 0,15g 20 µl 37,49 ng/µl
    pSB1C3_cut 0,08g 20 µl 4,56 ng/µl


    • Digestion of PCR product and vector:


    components PCR product /µl vector /µl
    DNA 187,58
    BSA (10x) 0,3 (100X used) 2
    Buffer 4 (10x)2,5 2
    Enzyme XbaI 1 1
    Enzyme PstI 1 1
    H2O2,2 6,42
    Total volume (e.g. 15,20,25,30 µl) 2520


    Ligation with T4-Ligase and transformation with BL 21 was made. Clones have to be picked tomorrow.

    Transformation evaluation of XL1B cells and repetition of transformation

    Investigator: Kira
    Against our expectations both agar plates contain very few colonies. In order to figure out if the lack of colonies is due to the XL1 blue cells or loss of pUC activity, test transformation will be repeated this evening with recently prepared XL1 blue cells as well as with Lab-XL1B cells. 50 pg pUC-plate contains 8 colonies 100 pg pUC-plate contains 18 colonies Transformation will be performed again with 100 pg pUC (= 2 ㎕ pUC).

    Cloning of SDM SspI and SDM PvuII

    Investigator: Chris W.

    • Vector: name: pSB1C3_SDM_SspI P125
    • Insert: name: pSB1C3_SDM_PvuII P129
    • new vector name: pSB1c3_SDM_SspI/PvuII P157
    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 144) BpmI ; Enzyme 2 (no.Lab: 152) ClaI
    • DNA concentration (vector): 321,9 ng/µl ; DNA concentration (insert): 308,4 µg/µl


    components volume of pSB1C3_SDM_SspI /µl volume of pSB1C3_SDm_PvuII /µl
    DNA 4,66 4,87
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme BpmI (no.Lab:144)1 1
    Enzyme ClaI (no.Lab:152)1 1
    H2O9,34 9,13
    Total volume (e.g. 15,20,25,30 µl) 2020

    Comment: I guess enzyme 144 was BpmI and enzyme 152 was ClaI ? (Jessica)



    Comment: u r right, changed (Chris)



    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45


    Loading plan for agarose gel:
    Marker used: GeneRuler ladder mix (Fermentas)

    Marker Sample 125, 20µl Sample 129, 20µl
    Lane 1 5 7


    Gel1.png


























    The mutual vector (now without SspI) and insert (now without PvuII) were cut out. The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

    • P125 Sspl: 12,8 ng/µl
    • P129 PvuII: 11,6 ng/µl


    The Ligation was performed as following:

    • Vector Volume: 2,81 µl
    • Insert Volume: 5,19 µl


    • 1µl T4-Ligase buffer (10x)
    • 8µl (Vector + Insert) mix
    • 1µl T4-Ligase


    Incubate for 30min


    Transformation: performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol

    Miniprep of p158 and p159

    Investigator: Bea & Volker

    Plasmid Mini-Prep

    For the tripple mutant of pAAV-RC a Quick-change reaction was carried out to remove the last restriction site (SalI) that is required for the Viral Brick standard. For this construct glycerol stocks and minipreps were carried out for two clones.


    Glycerol Stocks

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    Plasmidname pAAV_RC_1.2 SDM SalI align="left"| pAAV_RC_1.2 SDM SalI
    Date 05.08.2010 05.08.2010
    given glycerol-stock no. B126 B127
    given plasmid no. p158 p159


    TO do: Test digestion and sequencing!!!

    81.Labortag 06.08.2010

    Cloning Rep40 & Rep52 into pSB1C3

    Investigator: Patrick
    Intention: get biobrick ready
    PCR of pKEX-2XL.Rep 40 (P22) and pKEX-2XL.Rep 52 (P23) following gelrun (1%) and gelextraction.
    Used Primers:

    • Praefix Rep40_52ex (O94)
    • Suffix Rep 40_68ex (O96)
    • Suffix Rep 52_78ex (O97)


    PCR programm of Rep40:

    • 98°C 1 min


    • 98°C 15 sec
    • 63°C 25 sec
    • 72°C 15 sec Repeat this cycle 8 times.


    • 98°C 15 sec
    • 68°C 25 sec
    • 72°C 15 sec Repeat this cycle 17 times.


    • 72°C 5 min
    • Hold 4°C



    PCR programm of Rep52:

    • 98°C 1 min


    • 98°C 15 sec
    • 62°C 25 sec
    • 72°C 20 sec Repeat this cycle 8 times.


    • 98°C 15 sec
    • 66°C 25 sec
    • 72°C 20 sec Repeat this cycle 17 times.


    • 72°C 5 min
    • Hold 4°C


    ingredientsRep 40Rep40 + DMSORep52 Rep52 + DMSO
    5x Phusion HF buffer10 µl 10 µl10 µl10 µl
    10 mM dNTP mix1 µl 1 µl1 µl1 µl
    for Primer O94 (1:10 dilution, 05 µM)2,5 µl 2,5 µl2,5 µl2,5 µl
    rev Primer (1:10 dilution, 0,5 µM) 2,5 µl O962,5 µl O962,5 µl O972,5 µl O97
    DNA template 1 µl, 112,7 ng/µl1 µl, 112,7 ng/µl0,9 µl, 142,3 ng/µl0,9 µl, 142,3 ng/µl
    DMSO0 µl0,5 µl0 µl0,5 µl
    Phusion Polymerase0,5 µl0,5 µl0,5 µl0,5 µl
    H2O32,5 µl32 µl32,6 µl32,1 µl
    Total volume50 µl50 µl50 µl50 µl


    Ecpected size of Rep40: 940 bp, expected size of Rep52:1198 bp. There are two samples of Rep40 and Rep52 and the PCR of one of each was run with 1% DMSO because the Rep40 Praefix has a strong secondary structure and was used as a primer for Rep 40 and Rep 52

    IM000026 Patrick.JPG



    Digestion of pSB1C3 (P51.1) with EcoRI-HF and SpeI following gelrun (0.8%) and gelextraction.
    Expected size of the fragments: about 2000 bp and 800bp

    IM000028 Patrick.JPG



    All marked fragments were extracted. The Gelextraction was performed according to the standard protocol following a digestion of the PCR products with EcoRI HF and SpeI and a purification of the PCR product. The Ligation was not performed according to the standard protocol: 1 µl T4 DNA Ligase, 1 µl (10x) Buffer, 3 µl vector pSB1C3 (60 ng/µl) and 5 µl insert (all concentrations about 100 ng/µl). The Transformation (with BL21) was performed according to the standard protocol. Tomorrow there will hopefully be some clones on the agar plates.

    BioBrick Assembly of pSB1C3_beta globin_mVenus

    Investigator: Bea

    Comments: BioBricks are ready to use. The sequenced pSB1C3_beta globin (P133) and pGA14_mVenus (P60) will be digested with different enzymes and will be assembled in order to produce the first step in assembling the whole vector together.


    • First the plasmids P133 and P60 were digested:
      pSB1C3_betaglobin/µL pGA14_mVenus/µL
    DNA 7 9
    BSA (100x) 0,2 0,2
    Buffer 4 (10x) 2 2
    Enzyme XbaI 1 1
    Enzyme AgeI 1 1
    H2O 8,8 6,8
    Total volume 20 20
    • Incubation of plasmids at 37°C for 2 hours
    • Load samples on preparative 1% agarose gel and run at 110 V, 45 minutes


    Results: As it can be seen in the picture below, digestion of the vector pSB1C3_betaglobin (left lane) revealed a band at around 2500 - 3000 bp. The expected size after digetsing with SpeI and PstI was: 2555 bp. The band ran a little bit higher than expected, anyhow the band was cut out of the gel. The right lane belongs to the pGA14_mVenus which was digested with XbaI and PstI which resulted in two fragments. mVENUS was digested. Expected sizes were: mVenus = 756bp and pGA14 = 2900bp. The expected sizes correspond to the bands seen in the picture below. The smaller fragment belonging to mVenus was cut out of the gel as well.

    Freiburg10 pSB1C3 betaglobin mVenus 06 08 2010.jpg



    After gel extraction has been performed, the concentrations of the two fragments were measured and we obtained following concentrations:

    • c(pSB1C3_betaglobin) = 15,62 ng/µL
    • c(mVenus) = 6,83 ng/µL


    For ligation the T4-Ligase were used.

    • v(pSB1C3_betaglobin) = 2,64 µL
    • v(mVenus) =5,36 µL
    • v(10xT4 Ligase buffer) = 1 µL
    • v(T4-Ligase) = 1 µL
    • Total volume = 10 µL


    Additionally a control ligation was performed.

    • v(pSB1C3_betaglobin) = 2,64 µL
    • v(H20) =5,36 µL
    • v(10xT4 Ligase buffer) = 1 µL
    • v(T4-Ligase) = 1 µL
    • Total volume = 10 µL
    • Ligation duration was 45 minutes on room temperature.


    After ligation a transformation was performed.

    • Used bacterial strain: XL1-B
    • LB-agar plates containing chloramphenicol were plated and put in the 37°C room over night


    To do: Mini-Prep and test digestion in order to verify assembly of beta globin and YFP. After verification of the fusion of the two fragments this construct is ready for the next BioBrick assembly step.

    Test digestion of SalI SDM

    Investigator: Hanna

    Test digestion

    • buffer used: 3 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 53): SalI ; Enzyme 2: XbaI
    • Plasmid
      • Given Plasmid-Number: P158; DNA concentration: 398.8 ng/µL ;
      • Given Plasmid-Number: P159; DNA concentration: 437.4 ng/µL ;



    Components P158 Volume/µL P159 Volume/µL
    DNA 2.5 2.3
    BSA (10x) 1 1
    Buffer 3 (10x) 1 1
    SalI (no. Lab: 53) 0.5 0.5
    XbaI 0.75 0.75
    H2O 4.25 4.45
    Total volume 10 10


    Incubation: 1 h

    Agarose-Gel:


    0.5 g Agarose, 50 mL TBE (1 %),3 µL GELRED, at 115 Volt, running time: 45 minutes

    Sample Sample/µl] Loading dye (5x)/µl Expected size 1 (if SDM didn't work) Expected size 2 (if SDM didn't work) Expected size 3 (if SDM didn't work)
    P158 10 µl 2 µl 6129 bp 1143 bp 61 bp
    P159 10 µl 2 µl 6129 bp 1143 bp 61 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample P158 /µl Sample P159 /µl Marker /µL
    Lane 6.5 10 10 7


    Freiburg10 Test digestion SalI1.jpg

    Comments: Test digestion looked good: No 1143 bp fragment was detectable in neither test digestion.
    P158 was sent for sequencing to GATC.

    ITR test digestion of pAAV_MCS

    Investigator: Hanna

    Comment: Because sequencing of the right ITR delivered that perhaps a 15 bp fragment is missing in the sequence, we wanted to check, whether this fragment is also not present in the original pAAV_MCS plasmid or whether the loss of these 15 bp happened during cloning.


    Test digestion

    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1: NotI; Enzyme 2: PstI
    • Plasmid
      • Plasmid-Number: from Sven; DNA concentration: 259 ng/µL ;



    Components Volume/µL
    DNA 3.8
    BSA (10x) 1.5
    Buffer 4 (10x) 1.5
    NotI-HF 0.75
    PstI-HF 0.75
    H2O 3.2
    Total volume 15


    Incubation: 1 h

    Agarose-Gel:


    0.5 g Agarose, 50 mL TBE (1 %),3 µL GELRED, at 115 Volt, running time: 45 minutes

    Sample Sample/µl] Loading dye (5x)/µl Expected size ITRs Expected size 2 Expected size 3 Expected size 4
    pAAV_MCS 15 µl 2 µl 145 bp 1216 bp 555 bp 2608 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample /µl Marker /µL
    Lane 6.5 12 6.5


    Freiburg10 testDigestion pAAV MCS.jpg

    Comments: The test digestion showed that there're two bands between the 100 and 200 bp marker nucleotides! By comparing the gel picture with the last mini-prep digestion of the fancy method we found out that the space between the two bands are similar. Because of that we assumed that the 15 pb are already missing in the original Stratagene plasmid!!!


    Conclusion: Because we already tested the Stratagene Kit via YFP expression, we can conclude that the usage of the "wrong" rigth ITR works. Therefore we decided to continue working with this "mutated" = ENGINEERED  :) right ITR as BioBrick.


    Miniprep of pSBC13_Rep78, pSB1C3_Rep68, pSB1C3_AAP, pSB1C3_rightITR and psB1C3_leftITR

    Investigator: Kerstin

    Plasmid Mini-Prep



    Glycerol Stocks
    pSB1C3_Rep78

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B128 B129
    given plasmid no. P160 p161
    DNA-concentration ng/µl 139,1 71,0

    pSB1C3_2_Rep78

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B130 B131
    given plasmid no. P162 p163
    DNA-concentration ng/µl 112,1 79,7

    pSB1C3_Rep68

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B132 B133
    given plasmid no. P164 p165
    DNA-concentration ng/µl 80,2 72,8

    pSB1C3_2_Rep68

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B134 B135
    given plasmid no. P166 p167
    DNA-concentration ng/µl 85,6 69,1

    pSB1C3_AAP

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B136 B137
    given plasmid no. P168 p169
    DNA-concentration ng/µl 63,6 56,3

    pSB1C3_2_AAP

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B138 B139
    given plasmid no. P170 p171
    DNA-concentration ng/µl 135,1 114,2

    pSB1C3_rightITR

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B140 B141
    given plasmid no. P172 p173
    DNA-concentration ng/µl 73,7 66,4

    pSB1C3_leftITR

    Clone 1 Clone 2
    Bacteria strain BL-21 BL-21
    given glycerol-stock no. B142 B143
    given plasmid no. P174 p175
    DNA-concentration ng/µl 74,9 80,3


    Test digestion:
    pSB1C3_Rep78, pSB1C3_Rep68 and pSB1C3_AAP were digested with XbaI and SpeI
    pSB1C3_rightITR and pSB1C3_leftITR were digested with EcoRI and PstI.


    components volume of P160/µl volume of P161/µlvolume of P162/µl volume of P163/µl volume of P164/µlvolume of P165/µlvolume of P166µl volume of P167/µlvolume of P168µl volume of P169/µlvolume of P170/µlvolume of P171/µlvolume of P172/µlvolume of P173µl volume of P174/µl volume of P175/µl
    DNA 5,8 11,3 7,1 10 9,9 10,9 9,3 11,6 12,5 14,2 5,9 7,0 10,9 12,0 10,7 9,7
    BSA (10x) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
    Buffer 4 (10x)2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
    Enzyme 1 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5
    Enzyme 2 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5
    H2O 9,2 3,7 7,9 5,0 5,1 4,1 5,7 3,4 2,5 0,8 9,1 8 4,1 3 4,3 5,3
    Total volume /µl20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20


    Expected sizes:

    • Rep78: 1,8 kb
    • Rep68: 1,6kb
    • AAP: 650bp


    • left ITR: 150bp
    • right ITR: 150bp


    Results of samples loaded 1,5% agarose gel and ran ~50 minutes at 115 V:

    Freiburg10 test digestion itr und aap.jpg


    Results of samples loaded 0,8% agarose gel and ran ~50 minutes at 115 V:

    Freiburg10 test digestion rep 78 u. 68.jpg


    Sequencing

    P160, P165 and P170 were send for sequencing.

    82.Labortag 07.08.2010

    Cellculture

    Investigator: Patrick
    HEK 293 cells were split and seeded according to the standard protocol: 2x T75 flask and 10x10cm cellculture dishes.

    Continuation: Cloning Rep40 & Rep52 into pSB1C3

    Investigator: Patrick
    Results: no clones could be picked because there were no clones. Maybe the PCR purification or one of the following steps did not work (the PCR purifications were quite bad) so i purified the PCR products again yielding very low DNA concentrations (0-13,28 ng/µl) and still very obvious pollution of the sample. Nonetheless a ligation with the insert Rep40+DMSO, Rep52, Rep52+DMSO and the vector pSB1C3 was carried out following a transformation and according to the standard protocols. Hopefully there will be some clones tomorrow.

    Mini-Prep and test digestion of pSB1C3_SDM_SspI/PvuII

    Investigator: Jessica

    Plasmid Mini-Prep

    • new vector name: pSB1C3_SDM_SspI/PvuII


    Glycerol Stocks
    pSB1C3_SDM_SspI/PvuII

    Clone 1 Clone 2 Clone 3
    Bacteria strain BL21 BL21 BL21
    Plasmidname pSb1C3_SDM_SspI/PvuII pSb1C3_SDM_SspI/PvuII pSb1C3_SDM_SspI/PvuII
    Date 7.8.10 7.8.10 7.8.10
    given glycerol-stock no. B144 B145 B146
    given plasmid no. P157.1 P157.2 P157.3



    Test digestion

    • buffer used: 2 ; Restriction-enzymes used: Enzyme 1 SspI ; Enzyme 2 PvuII
    • Plasmid: pSB1C3_SDM_SspI/PvuII:
      • Given Plasmid-Number: P157.1; DNA concentration: 246,3 ng/µL ;
      • Given Plasmid-Number: P157.2; DNA concentration: 231,7 ng/µL ;
      • Given Plasmid-Number: P157.3; DNA concentration: 219,6 ng/µL ;


    Components P157.1 Volume/µL P157.2 Volume/µL P157.3 Volume/µL
    DNA 2,8 3,0 3,19
    BSA (10x) - - -
    Buffer no. 2 (10x) 1 1 1
    Enzyme 1 SspI 0.5 0.5 0.5
    Enzyme 2 PvuII 0.5 0.5 0.5
    H2O 5,2 5 4,81
    Total volume 10 10 10


    • Incubation: 45 min


    Agarose-Gel:


    0.45 g Agarose, 50 mL TAE, 3 µL GELRED, at 115 Volt, running time: 45 minutes

    Sample Sample/µl] Loading dye (6x)/µl
    P157.1 10 µl 1,6 µl
    P157.2 10 µl 1,6 µl
    P157.3 10 µl 1,6 µl


    • Marker: GeneRuler ladder mix
    Marker /µL Sample P157.1 /µL Sample P157.2 /µL Sample P157.3 /µL
    Lane 7 10 10 10



    I expect one cut from the PvuII RS in the CFP --> linearized

    Prep pSB1C3 SDM SspIPvuII.jpg


    Comment:

    Comments:Oh f... it makes no sense... if the cloning didn't work, there can just be 3 bands (one time SspI and twice PvuII). The results of the test digestion from aug 1th showed that there can be one RS PvuII more. but then you have a maximum of 4 bands. P157.2 show 5 bands. i can't interpret this result at the moment. interpretation will follow




    Evaluation of test transformation with XL1 blue cells and pUCI

    Investigator: Kira

    Transformation was performed with lab XL1B and iGEM XL1B cells. iGEM plate contained just 25 colonies, while lab plate contained around 50 colonies. Conclusion: the production of competent cells has to be repeated.

    Biobrick production of p5 promoter from pTAV2 and pAAV_RC

    Investigator: Kira

    • PCR:

    DNA samples were diluted 1:100


    Ingredients p 32 p 50
    5X Phusion HF buffer 10 µl10 µl
    10 mM dNTP mix1µl 1 µl
    forward primer: O114 2,5µl2,5 µl
    reverse primer: O115 2,5 µl2,5 µl
    DNA Template0,3 µl 0,4 µl
    DMSO 0 µl0 µl
    Phusion Polymerase0,5 µl 0,5 µl
    H2O33,2 µl28,6 µl
    Total volume50 µl 50 µl


    PCR program:

    CyclesTemperatureTime
    98°C1
    8x98°C30"
    60°C25"
    72°C6"
    17x98°C30"
    70°C25"
    72°C6"
    1x72°C5'
    Hold 4°C


    Digestion of plasmid backbone:

    c (pSB1C3) = 151, 1 ng/ µl

    Components vector Volume/µL
    DNA 1 µg 6,0 µl
    BSA (100x) 0,2 µl
    Buffer no. 4 (10x) 2,0 µl
    Enzyme 1 XbaI 1 µl
    Enzyme 2 PstI HF 1 µl
    H2O 9,8 µl
    Total volume 20


    incubation @ 37 C for approx. 2 h

    1% agarose gel with PCR products:

    Freiburg10 PCR.jpg


    Concentrations after gel extraction:

    c(p32) = 67,95 ng/µl
    c(p50) = 32,45 ng/µl
    c(vector) = 7,54 ng/µl

    Digestion of PCR products:

    Components PCR product Volume/µL
    DNA 1 µg 23,0 µl
    BSA (100x) 0,3 µl
    Buffer no. 4 (10x) 3,0 µl
    Enzyme 1 XbaI 1,5 µl
    Enzyme 2 PstI HF 1,5 µl
    H2O 0,7 µl
    Total volume 30


    Concentrations after PCR purification:

    c(p32) = 21, 63 ng/µl
    c(p50) = 19,48 ng/µl

    Ligation:

    Quickligase was used for ligation.

    p32 DNA-Mix: 8 µl vector + 1 µl insert
    p50 DNA-Mix: 7,9 µl vector + 1,1 µl insert

    Transformation:

    Transformation was performed according to the standard protocol with DYT and BL21 cells. The cells were spread on the agar plates containing chloramphenicol.

    Sequencing results of SalI SDM

    Investigator: Hanna

    Comment: The test digestion already showed that the SDM of the SalI restriction site in pAAV_RC worked. This could be validated by sequencing the plasmid with the Rep-1250_for primer:


    Freiburg10 SalI.jpg



    P58 can be used for further RepCap-experiments.

    83. Labortag 08.08.2010

    HT 1080 cells were split and seeded: 3x10^6 cells per into each well of a 6-well plate.

    A431 cells were washed and received new medium.

    There were no clones on the agar plates so they were put back into the 37°C room. Maybe there will be some clones tomorrow.

    84. Labortag 09.08.2010

    Midi-Prep Inoculation

    Investigators: Chris W., Patrick

    • pAAV_RC
    • pAAV_RC_1.2 SDM SalI (P158)
    • pAAV_RC_mGMK_TK30 clone 1 (P81)
    • pAAV_RC_mGMK_TK30 clone 2 (P82)

    Continuation: Cloning Rep40 & Rep52 into pSB1C3

    Investigator: Patrick
    Two Rep52+DMSO clones grew. They were picked and tomorrow there will be a midi prep and a test digestion. Simultaneously a new approach for cloning Rep40 and Rep52 into pSB1C3 was performed. (see next header)

    Repetition: BioBrick production of Rep40 & Rep52

    Investigator: Anna

    Comments: Another approach was done because there were no clones for Rep 40 and only two for Rep 52 (Cloning see 06.08.2010). PCR was performed again with DMSO. Samples from gel extraction are stored in 4°C freezer.



    PCR

    Ingredients Rep 40 Rep52
    5X Phusion HF buffer 10 µl10 µl
    10 mM dNTP mix1µl 1 µl
    forward primer: O094 2,5µl2,5 µl
    reverse primer: O0096/ O097 2,5 µl2,5 µl
    DNA Template (1ng)1 µl 1 µl
    DMSO 1 µl1 µl
    Phusion Polymerase0,5 µl 0,5 µl
    H2O31,5 µl31,5 µl
    Total volume50 µl 50 µl


    PCR programm

    PCR Programtemperature/ °CTime PCR products
    198 1min
    298 15s
    8x*** 15s63°C
    3 72***15s
    4 98 15s
    19x***15s68°C
    572***15s
    6x725min
    Hold4


    Agarose gel

    0.5 g Agarose, 50 mL TAE (1 %),3 µL GELRED, at 120 Volt, running time: 45 minutes

    Sample Sample / µl] Loading dye (6x) / µl Expected size
    p22 (Rep40) 44 µl 7 µl 940 bp
    p23 (Rep52) 44 µl 7 µl ~1200 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample Rep40 /µl Sample Rep52 /µl
    Lane 7 51 51


    Freiburg10 Rep40 Rep52.png

    Gel extraction
    PCR products were eluted with 20 µl BE puffer following the standard protocol.
    c(Rep40)= 28,03 ng/µl
    c(Rep52)= 121,16 ng/µl

    Comments: Digestion was done with the wrong enzymes (EcoRI HF and SpeI), it will be repeated on 11.08. with XbaI and SpeI


    Sequence analysis of pSB1C3_mGMK

    Investigator: Bea

    Comments: mGMK was cloned into the pSB1C3 backbone in order to send it to the parts registry!. The sequence analysis revealed that the mGMK was successfully cloned into pSB1C3 plasmid. This polasmid is in the RFC25 standard.

      Results sequencing: Sequence read ok!
      Primer used: VR-2
      Comment: A mutation has been found in the pSB1C3 backone which was found generally in the backbone which we received from the iGEM HQ.
    Freiburg10 Sequence analysis of pSB1C3 mGMK 09 08 2010.jpg

    Mini-Prep of pSB1C3_hTERT, pSB1C3_beta-globin_mVenus, pSB1C3_pTAV2(P5), pSB1C3_pAAV_RC(P5TATAles)

    Investigator: Jessica

    Bold text Mini-Prep following the standart Protokoll

  • P177 = pSB1C3_hTERT clone1 = 163,6 ng/µl
  • P178 = pSB1C3_hTERT clone2 = 312,0 ng/µl
  • P179 = pSB1C3_betaglobin_mVenus clone1 = 237,2 ng/µl
  • P180 = pSB1C3_betaglobin_mVenus clone2 = 228,2 ng/µl
  • P181 = pSB1C3_pTAV2(P5) clone1 = 108,8ng/µl
  • P181 = pSB1C3_pTAV2(P5) clone2 = 140,9 ng/µl
  • P181 = pSB1C3_pAAV_RC(P5TATAless) clone1 = 165,0 ng/µl
  • P181 = pSB1C3_pAAV_RC(P5TATAless) clone2 = 176,6 ng/µl
  • P181 = pSB1C3_pAAV_RC(P5TATAless) clone3 = 176,9 ng/µl


    Test digestion:
    • Perfomed with 10 µL of total volume with all 9 clones
    •   Mastermix/µL P176/µL P177/µL P178/µL P179/µL P180/µL P181/µL P182/µL P183/µL P184/µL
      DNA - 4,3 2,3 3,0 2,4 6,4 5,0 4,2 4,0 4,0
      BSA (10x) 10 3 3 3 3 3 3 3 3 3
      Buffer 4 (10x) 10
      Enzyme XbaI 5
      Enzyme AgeI 5
      H2O - 2,7 4,7 4,0 4,6 0,6 2,0 2,8 3,0 3,0
      Total volume 30 10 10 10 10 10 10 10 10 10


    • All 9 samples were loaded on a 1% agarose gel
    • P176 = pSB1C3_hTERT clone1
    • P177 = pSB1C3_hTERT clone2
    • P178 = pSB1C3_betaglobin clone1
    • P179 = pSB1C3_betaglobin clone2
    • P180 = pSB1C3_pTAV2(P5) clone1
    • P181 = pSB1C3_pTAV2(P5) clone2
    • P182 = pSB1C3_pAAV_RC(P5TATAless) clone1
    • P183 = pSB1C3_pAAV_RC(P5TATAless) clone2
    • P184 = pSB1C3_pAAV_RC(P5TATAless) clone3


    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 (Geneious)
    P176 10 µl 1,6 µl 474 bp
    P177 10 µl 1,6 µl 474 bp
    P178 10 µl 1,6 µl 509 bp
    P179 10 µl 1,6 µl 509 bp
    P1780 10 µl 1,6 µl 164 bp
    P181 10 µl 1,6 µl 164 bp
    P182 10 µl 1,6 µl 164 bp
    P183 10 µl 1,6 µl 164 bp
    P184 10 µl 1,6 µl 164 bp
    9 Proben tert, beta, p5.jpg



    Comments: hTERT and betaglobin_mVenus is inserted in pSB1C3, pTAV(P5) and pAAV_RC (P5TATAless) will be sequenced


    Primer: Reverse Primer VR2

    Cell culture

    Investigators: Adrian, Kerstin, Patrick

    Transfection on HEK293


    The motivation: first we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression).

    Plate pAAV_RC/µgpHelper/µgGOI/µg
    13,3 3,3 no GOI
    26,6 3,3 no GOI
    33,3 3,3 3,3 TK_GMK clone1
    43,3 3,3 3,3 TK_GMK clone1
    53,3 3,3 3,3 TK_GMK clone2
    63,3 3,3 3,3 YFP
    73,3 3,3 10 YFP
    83,3 3,3 20 YFP
    93,3 3,3 40 YFP
    103,3 3,3 60 YFP
    Transduction of HT1080


    Motivation The Motivation: checking survivalrate of Cells, with either no Virus, virus without GOI and virus with YFP (10µg stock)
    The Plan:
    Plate I

    control, no cells no GOI 150µl10µg 500µl|
    control, no virusno GOI 300µl 10µg 750µl|


    Plate II

    control, no cells no GOI 150µl10µg 500µl|
    control, no virusno GOI 300µl 10µg 750µl|

    Plate III

    control, no cells no GOI 150µl10µg 500µl|
    control, no virusno virus 10µg 750µl|
    Passaging and seeding of A431

    one 6er dish was prepared for making pictures and one T75 Flask.


    BioBrick Assembly of pSB1C3_leftITR_CMV and pSB1C3_hGH_rightITR

    Investigator: Bea, Achim, Jo

    Comments: In order to proceed with the BioBrick assembly the pasmids pSB1C3_left ITR and pSB1C3_CMV were used and pSB1C3_rigthITR and pSB1C3_hGH were used in order to obatin plasmids with both sequences.



    Digestion:

    components pSB1C3_left ITR pSB1C3_CMV pSB1C3_right ITR pSB1C3_hGH
    DNA 13,4 9,5 13,6 6,6
    BSA (10x) 2 2 2 2
    Buffer 4 (10x)2 2 2 2
    Enzyme1 1 (SpeI) 1 (Xba1)1 (EcoRI) 1 (EcoRI)
    Enzyme2 1 (PstI)1 (PstI) 1 (XbaI)1 (SpeI)
    H2O0,6 4,5 0,4 7,4
    Total volume 2020 2020


    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes


    Freiburg10 9 8 2010 Digestion of psB1C3 rITR psB1C3 lITR psB1C3 CMV psB1C3 hGH125.jpg

    Expected sizes of constructs:

    • pSB1C3_CMV: 650 bp
    • pSB1C3_lITR: 2200 bp
    • pSB1C3_hGH: 500 bp
    • pSB1C3_rITR: 2200 bp

    The correspnding bands were cut out and Gel-Extraction was performed according to protocol.





    concentrations measured via NanoDrop:

    • pSB1C3_CMV: 13,51 ng/µl
    • pSB1C3_lITR: 18,49 ng/µl
    • pSB1C3_hGH: 7,22 ng/µl
    • pSB1C3_rITR: 20,24 ng/µl


    Ligation:
    For ligation, T4 ligase was used.

    • 1µl T4-Ligase buffer (10x)
    • 8µl (Vector + Insert) mix
    • 1µl T4-Ligase

    Ligation was carried out as following:

    • Ligation 1:
      • pSB1C3_CMV: 4,4 ng/µl
      • pSB1C3_lITR: 3,6 ng/µl
    • Ligation 2:
      • pSB1C3_hGH: 5,25 µl
      • pSB1C3_rITR: 2,75 µl



    Incubation for 40 minutes.


    Transformation:
    Transformation carried out according to standard protocol (BL21). Cells were plated on an agar plate with chloramphenicol.

    Repetition: Cloning of SDM SspI and SDM PvuII

    Investigator: Stefan

    Comments: Last week's cloning of SDM_SspI and SDM_PvuII didn't work out, so this is the repetition. In this approach the same restriction enzymes and amounts of DNA were used.



    • Vector: name: pSB1C3_SDM_SspI P125
    • Insert: name: pSB1C3_SDM_PvuII P129
    • buffer used: 4
    • Restriction-enzymes used:
      • BpmI (no. Lab: 144)
      • ClaI (no.Lab: 152)
    • DNA concentration (P125): 321,9 ng/µl
    • DNA concentration (P129): 308,4 ng/µl


    Digestion:

    components volume of pSB1C3_SDM_SspI /µl volume of pSB1C3_SDm_PvuII /µl
    DNA 4,66 4,87
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    BpmI (no.Lab:144)1 1
    ClaI (no.Lab:152)1 1
    H2O9,34 9,13
    Total volume 2020


    Gel extraction:
    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

    Expected sizes of constructs:

    • pSB1C3_SDM_SspI: 1819 bp
    • pSB1C3_SDM_PvuII: 981 bp

    The correspnding bands were cut out and Gel-Extraction was performed according to protocol.

    Freiburg10 pSB1C3 SDM SspI and PvuII 090810.png

















    concentrations measured via NanoDrop:

    • Sspl: 19,85 ng/µl
    • PvuII: 10,57 ng/µl


    Ligation:
    For ligation, T4 ligase was used.

    • 1µl T4-Ligase buffer (10x)
    • 8µl (Vector + Insert) mix
    • 1µl T4-Ligase

    The Ligation was performed as following:

    • SDM_SspI Volume: 4,03 µl
    • SDM_PvuII Volume: 3,97 µl


    • 1µl T4-Ligase buffer (10x)
    • 8µl (Vector + Insert) mix
    • 1µl T4-Ligase


    Incubating for 45 minutes.


    Transformation:
    Transformation performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol.

    Design and ordering of primer for P40 Promoter BioBrick production

    Investigator: Hanna

    In order to generate a P40 promoter BioBrick = "Cap-Promoter", oligos were designed and ordered at Sigma Aldrich:

    Freiburg10 P40.jpg


    85. Labortag 10.08.2010

    Retrafo with RepCap gene (Mr.Gene)

    Investigator: Bea

    Comments: We received the rep_cap insert ordered from Mr. Gene today. In order to used and clone the insert into the desired sequences a retrafo has to be performed.

    Re-transformation:

    • Construct used: pMA_REP_CAP
    • We reveived 5 µg (lyophilzied). These were resuspended in 100 µL H2O and vortexted.
    • c = 50 ng/µL
    • c = Plasmid need to be diluted in order to obatain the final 500pg
    • Dilution: 1:100
    • 100 µL aliquot of XL1-B were thawed on ice. The proper amount of 1µL was added to the cells.
    • Transformed cells were plated on LB plates containing ampicillin

    Continuation: Cloning Rep40 & Rep52 into pSB1C3

    A midi prep of the two Rep52+DMSO clones was performed yielding concentrations of 261,35 ng/µl (clone 1) and 240,47 ng/µl (clone 2). Unfortunately we noticed today that we should not use EcoRI to clone Rep52 into pSB1C3 because EcoRI cuts two times within the sequence of Rep52.

    Midi-Prep of pAAV_RC, pAAV_RC_1.2 SDM SalI (P158), pAAV_RFC25_mGMK_TK30 clone 1 (P81) and 2 (P82)

    Investigators: Patrick, Chris W.

    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    pAAV_RC P81aP81bP82aP82bP158aP158b
    1348,49 ng/µl1783,86 ng/µl 2096,25 ng/µl1915 ng/µl1469,35 ng/µl1834,25 ng/µl1771,27 ng/µl


    The annotations a and b were added because two midi-preps of P81, P82 and P158 were carried out.

    Results of sequencing pSB1C3_pTAV2(P5) and Psb1C3_pAAV_RC(P5TATAless)

    Investigator: Jessica

    Mistake.png

    @Jessi: Which clones are ok, which not?? text does not correspond to pictures! (Bea)

    Freiburg10 Sequence alignment P5 mit TATA clone1neu.jpg














    sequencing of P180 was successful, P5 with TATAbox is in pSB1C3

    Freiburg10 Sequence alignment P5TATAless clone2neu.jpg















    sequencing of P183 was successful but there is a G missing --> plamid is thrown away

    Freiburg10 Sequence alignment P5TATAless clone3.jpg













    sequencing of P184 was successful, P5 is in without TATAbox. this plasmid can be used.

    P181 and P180 are thrown away because of the result of the test digestion.

    Repetition: BioBrick production of Rep40 and Rep 52

    Investigator: Anna

    Comments: PCR was repeated once again (that was bad!), because digestion was done with the wrong enzymes (EcoRI HF and SpeI). There are some differences in the PCR programm in comparison to the last (see 09.10.)



    PCR

    Ingredients Rep 40 Rep52
    5X Phusion HF buffer 10 µl10 µl
    10 mM dNTP mix1µl 1 µl
    forward primer: O094 2,5µl2,5 µl
    reverse primer: O0096/ O097 2,5 µl2,5 µl
    DNA Template 1:100 (1ng)1 µl 1 µl
    DMSO 1 µl1 µl
    Phusion Polymerase0,5 µl 0,5 µl
    H2O31,5 µl31,5 µl
    Total volume50 µl 50 µl


    PCR programm

    PCR Programtemperature/ °CTime PCR products
    198 1min
    298 15s
    8x*** 15s62,5°C
    3 72***25s
    4 98 15s
    19x***15s67°C
    572***25s
    6x725min
    Hold4


    Agarose gel

    0.5 g Agarose, 50 mL TAE (1 %),3 µL GELRED, at 120 Volt, running time: 45 minutes

    Sample Sample / µl] Loading dye (6x) / µl Expected size
    p22 (Rep40) 50 µl 8 µl 940 bp
    p23 (Rep52) 50 µl 8 µl ~1200 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample Rep40 /µl Sample Rep52 /µl
    Lane 7 58 58


    Freiburg10 Rep40 52.jpg
































    Comments: Digestion of the samples will be done tomorrow with XbaI and SpeI.



    Gelextraction

    Elution was done with 20 µl Pe buffer following the standard protocol.

    c(Rep40)= 23,1 ng/µl
    c(Rep52)= 78,0 ng/µl

    Preparation of competent XL1blue and testtrafo

    Investigator: Jessica
    preparation of competent E.coli XL1blue was made according to the standardprotocol (91 eppis with 60µl) they are in the green boxes in -80°C
    testtrafo was made

    • one plate with 50pg/µl
    • one plate with 100pg/µl

    ...trafo is failed because amp was missing on the plates (resistence in pUC). trafo will be repeated on aug 11th by Tobi...

    Repetition: Mini-Prep and test digestion of pSB1C3_SDM_SspI/PvuII

    Investigator: Adrian, Stefan

    Plasmid Mini-Prep

    • vector name: pSB1C3_SDM_SspI/PvuII


    Glycerol Stocks
    pSB1C3_SDM_SspI/PvuII Only clone 1 grew, therefore there is just one stock = B159


    Test digestion

    • buffer used: 2
    • Restriction-enzymes used:
      • Enzyme 1 SspI
      • Enzyme 2 PvuII
    • Plasmid: pSB1C3_SDM_SspI/PvuII:
      • Plasmid-Number: P185; DNA concentration: 47,9 ng/µL

    comment: DNA was eluted into previously used tube, therefore contamination is possible.




    Components P185 Volume/µL P51.1 Volume/µL
    DNA 18,1 6,8
    BSA (10x) 2,5 2,5
    Buffer no. 2 (10x) 2,5 2,5
    Enzyme 1 SspI 0.5 0.5
    Enzyme 2 PvuII 0.5 0.5
    H2O 0,9 12,2
    Total volume 10 10 |


    • Incubation: 45 min


    Agarose-Gel:


    0.4 g Agarose, 50 mL TAE, 3 µL GELRED, at 115 Volt, running time: 45 minutes


    • Marker: GeneRuler ladder mix
    Marker /µL Sample P185 /µL Sample P51.1 /µL
    Lane 7 17 17


    One cut is expected (PvuII in CFP).

    Freiburg10 SspI PvuII test digestion.png


    Comment: There are 6 bands now. This test digestion again looks very different from that of P157.2 done by Jessica (lab day 82). Results should be discussed tomorrow.



    Picking clones of pSB1C3_SDM_SspI/PvuII

    Investigator: Stefan

    Comments: Test digestion didn't work out, three additional clones were picked from the plate we get from yesterday's Trafo to retry tomorrow.

    • Bacterial strain used: BL21
    • Inoculating of 10 mL DYT medium containing 10µL chloramphenicol
    • Put in 37°C room on shaker at 20.45hours.


    86. labday 11.08.2010

    Transduction of HT1080 with no AAV, AAV without GOI and AAV with 10µg

    The motivation: In the past, the percentage of living cells in FACS was very low (~ 70%) so we want to check if the AAV-induced stress is responsible for cellular death, or the detaching procedure (trypsin and mechanical stress).
    The plan
    Plate I

    control, no cells no GOI 150µl10µg 500µl
    control, no virusno GOI 300µl 10µg 750µl


    Plate II

    control, no cells no GOI 150µl10µg 500µl
    control, no virusno GOI 300µl 10µg 750µl

    Plate III

    control, no cells no GOI 150µl10µg 500µl
    control, no virusno virus 10µg 750µl


    Biobrick Assembly:Picking Clones, Miniprep, Testdigestion of CMV&lITR/ hGH & rITR

    Investigator: Chris L., Achim
    Test Digestion showed that the cloning was succesful. Freiburg10 assembly1.jpg

    NanoDrop concentrations:

    • hGH_rITR1:105,15 ng/µl
    • hGH_rITR2:96.97 ng/µl
    • CMV_lITR1:231.61 ng/µl
    • CMV_lITR2:129.19 ng/µl


    Biobrick Assembly: bSB1C3_lITR_CMV & pSB1C3_betaglobin_mVenus

    Investigator: Chris L., Achim

    Comments: In order to proceed with the BioBrick assembly the plasmids pSB1C3_leftITR_CMV was cut with SpeI and PstI, pSB1C3_betaglobin_mVenus was cut with XbaI and PstI. The insert fragment containing betaglobin_mVenus was ligated to the vector fragment containing the leftITR_CMV promotor.



    Digestion:

    components pSB1C3_leftITR_CMV pSB1C3_betaglobin_mVenus
    DNA 5,2 8,4
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme1 1 (SpeI) 1 (Xba1)
    Enzyme2 1 (PstI)1 (PstI)
    H2O8,8 5,6
    Total volume 2020


    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

    Expected sizes of constructs:

    • pSB1C3_leftITR_CMV: 2800 bp
    • pSB1C3_betaglobin_mVenus: 1200 bp, 2000 bp

    The corresponding bands were cut out and Gel-Extraction was performed according to protocol.

    concentrations measured via NanoDrop:

    • pSB1C3_leftITR_CMV: 55,5 ng/µl
    • pSB1C3_betaglobin_mVenus:: 10,4 ng/µl


    Continuation of Cloning of Rep40/52

    Investigator: Anna


    • Digestion of PCR products and vector:


    components PCR product /µl vector /µl
    DNA 19 10
    BSA (10x) 3 3
    Buffer 4 (10x)3 3
    Enzyme XbaI 1 1
    Enzyme SpeI 1 1
    H2O3 12
    Total volume (e.g. 15,20,25,30 µl) 3030


    Comments: This time digestion was done with XbaI and SpeI (means it has to be checked if the inserts are cloned into the vector in the right orientation).


    • Purification of Rep40 and Rep52:

    For the purification 95 µl of buffer PBI was used.

    c(Rep40)= 12,5 ng/µl
    c(Rep52)= 51,8 ng/µl



    • Gelextraction of pSB1C3_RFC25_CFP:

    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time:55
    5µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)



    Digestion of pSB1C3 2.jpg


    c(pSB1C3)= 10,59 ng/µl
    c(pSB1C3_2)= 4,25 ng/µl


    • Ligation of PCR products and vector:

    For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used.

    Sample-no.vector /µl insert /µl
    pSB1C3 + Rep40 1 3,65 4,35
    pSB1C3 + Rep52 2 5,87 2,13
    Total volume (e.g. 15,20,25,30 µl) 8


    • Transformation:

    The transformation was done following the standard protocol using B21 cells.

    PCR of bla_14FM and ligation into pSB1C3_SDM_SspI and pSB1C3_SDM_PvuII for virobrick-production T


    Investigator: Anissa,Kerstin


    Comments: Because it still is unclear if SDM of pSB1C3_SspI_PvuII has worked, we decided to use the one time mutagenisised vector pSB1C3_SDM_SspI, to clone bla into it and to make the second mutation of PvuII after bla is inserted. .



    • PCR:

    (was performed following the standard protocol)

    Ingredients v(pTB106_bla14FM) - DMSO / µl v(pTB106_bla14FM) + DMSO / µl
    5X Phusion HF buffer 10 10
    10 mM dNTP mix1 1
    forward primer: O116(P extreme f)2,5 2,5
    reverse primer: O117 (P extreme r) 2,5 2,5
    DNA Template 1ng 5,3 µl 5,3 µl
    DMSO (1%) 0,5 0,5
    Phusion Polymerase0,5 0,5
    H2O28,2 27,7
    Total volume50 50


    • PCR program:


    PCR Programtemperature/ °C |time
    198 1'
    298 15
    8x61 25
    3 72 15
    4 98 15
    572 15
    1x72 5'
    Hold4


    Freiburg10 pcr bla14FM.JPG


    • Digestion PCR product:


    • DNA-Concentration: 4,2 ng/µl


    componentsvolume / µl
    DNA 28,5
    BSA 100x 0,4
    Buffer: 4 10x 4
    Enzyme 1 (XbaI) 1
    Enzyme 2 (PstI) 1
    H2O 5,1
    Total volume 40
    • Purification of PCR product: (QlAquick Gel Extraction Kit)


    preparation of purification was made according to the standardprotocol


    Comments: The wrong enzymes were used. Experiment has to be done tomorrow.


    Test digestion pSB1C3_CFP_SDM SspI and PvuII

    Investigator: Stefan

    • buffer used: 2; Restriction-enzymes used: Enzyme 1: ClaI ; Enzyme 2: BpmI ; Enzyme 3: SspI ; Enzyme 4: PvuII
    • Plasmids:
      • pSB1C3_SDM_SspI/PvuII P185 (first tree lanes, cut with different enzymes)
      • pSB1C3_RFC25_longlinker P105
      • pSB1C3_CFP P51.1


    Components P185/µL P185/µL P185/µL P105/µL P51.1/µL
    DNA 18 18 18 4,86,8
    BSA (10x) 2,5 2,5 2,5 2,5 2,5
    Buffer 2 (10x) - - - 2,5 2,5
    Buffer 4 (10x) 2,5 2,5 2,5 - -
    Enzyme 1 ClaI (152) 0,5 0,5 0,5 - -
    Enzyme 2 BpmI (144) 0,5 0,5 0,5 - -
    Enzyme 3 SspI (68) - 0,5 - 0,5 0,5
    Enzyme 4 PvuII (50) - - 1 0,5 0,5
    H2O 1 - 0,5 14,2 12,2
    Total volume 25 25 25 25 25


    • Incubation: 75 minutes

    Agarosegel
    0.5 g Agarose, 50 ml TAE, 3 µL GELRED, at 115 Volt, running time: 60 minutes



    Freiburg10 SspI PvuII ClaI BpmI later.png





























    comments: Test digestion of pSB1C3_SDM_SspI/PvuII with ClaI, BpmI and SspI looks the same as with ClaI and BpmI. Therefore we can assume that there is no additional restriction site within the vector. But still the bands above 3000 bp cannotz be explained. A new test digestion will be performed tomorrow to check the size of the vector.
    The approach cut with ClaI, BpmI and PvuII looks strange. Propably, PvuII does not work correctly. New PvuII-HF was ordered and delivered so this can be verified tomorrow.
    pSB1C3_RFC25_longlinker digested with SspI and PvuII seems to be not completely digested This could correspond to non-working PvuII. pSB1C3_CFP looks like expected.

    pSB1C3_SDM_Ssp1 (P125) sent for sequenzing


    Investigator: Stefan

    comment: To verify the correct deletion of the SspI restriction site it was sent for sequenzing.

    Primer used: GATC_std_pTeSp-2

    Picking clones of pSB1C3_SDM_SspI/PvuII (P185)


    Investigator: Adrian

    comment: pSB1C3_SDM_SspI/PvuII (P185) is empty. For plasmid production DYT + Cm was inoculated from the glycerol stock (B159).

    87. labday 12.08.2010

    Virus production, seeding A431 cell

    Investigator: Adrian

    In first place we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression).
    Ten viral stocks with 3 ml each were prepared.

    Plate pAAV_RC/µgpHelper/µgGOI/µg
    13,3 3,3 no GOI
    26,6 3,3 no GOI
    33,3 3,3 3,3 TK_GMK clone1
    43,3 3,3 3,3 TK_GMK clone1
    53,3 3,3 3,3 TK_GMK clone2
    63,3 3,3 3,3 YFP
    73,3 3,3 10 YFP
    83,3 3,3 20 YFP
    93,3 3,3 40 YFP
    103,3 3,3 60 YFP

    XL1blue are ready to use

    Investigator: Jessica
    there are 60µl aliquots to use for one trafo

    Mini-Prep of pSB1C3_SDM_SspI/PvuII and pMA_RC_insert


    Investigator: Stefan

    Gycerol stock was prepared for pMA_RC_insert and labeled B167. No glyerol stock for pSB1C3_SDM_SspI/PvuII because cells from earlier stock werer used for Mini-Prep.

    Miniprep was performed according to protocol.

    Samples were labeled:

    • pSB1C3_SDM_SspI/PvuII = P185.1
    • pMA_RC_insert = P190

    Concentrations:

    • pSB1C3_SDM_SspI/PvuII (P185.1): 257,2 ng/µl
    • pMA_RC_insert (P190): 339,5 ng/µl


    No test digestion was performed.

    Biobrick Assembly: pSB1C3_lITR_CMV & pSB1C3_betaglobin_mVenus

    Investigator: Chris L., Achim

    Comments: In order to proceed with the BioBrick assembly the plasmids pSB1C3_leftITR_CMV was cut with SpeI and PstI, pSB1C3_betaglobin_mVenus was cut with XbaI and PstI. The insert fragment containing betaglobin_mVenus was ligated to the vector fragment containing the leftITR_CMV promotor.


    Ligation:
    For ligation, T4 ligase was used.

    • 1µl T4-Ligase buffer (10x)
    • 8µl (Vector + Insert) mix
    • 1µl T4-Ligase

    Ligation was carried out as following:

    • pSB1C3_leftITR_CMV: 6,98 µl
    • pSB1C3_betaglobin_mVenus: 1,02 µl


    Incubation for 40 minutes.

    • Transformation:

    The transformation was done following the standard protocol using XL1B cells.


    Repetition of cloning of bla14FM_viralbrick_MCS into pSB1C3_SDM_SspI


    Investigator: Anissa

    Comments:Yesterday the pcr-product of bla14FM was cut with the wrong enzymes, so no overhangs could result. Today it will be repeated with the rihgt ones (SpeI and NgomIV) .


    • Digestion of the PCR product bla14FM for 2 h :


    • DNA-Concentration: 10,2 ng/µl


    componentsvolume / µl
    DNA 18
    BSA 100x 3
    Buffer: 4 10x 3
    Enzyme 1 (NgomIV) 1
    Enzyme 2 (SpeI) 1
    H2O 4
    Total volume 30
    • Purification of PCR product: (QlAquick Gel Extraction Kit)


    preparation of purification was made according to the standardprotocol

    • Digestion of vector backbone pSB1C3_SDM_SspI for 2 h:


    • 1 µg vector has been used
    componentsvolume / µl
    DNA 3,12
    BSA 100x 1,5
    Buffer: 4 10x 1,5
    Enzyme 1 (NgomIV) 1
    Enzyme 2 (SpeI) 1
    H2O 6,88
    Total volume 15


    • the cut vector was loaded on a preperative 1% agarose gel and the gel was running for 45 minutes


    Freiburg10 pSB1C3 SDM SspI cut.png


    • gel was cut and gelextraction was performed according the qiagen standard protocol.


    • Ligation of pSB1C3_SDM_SspI and bla14FM:


    componentsvolume / µl
    t4 buffer (10X) 1
    t4 Ligase 1
    vector DNA 2,72
    Insert 5,28


    • Transformation: pSB1C3_SDM_SspI_bla14FM was transformed into BL21 following the stanard protocol

    Quickchange of the EaglII restriction site in the PMA_RepCap vector

    Investigator: Chris L., Achim

    Comments: We performed a QuikChange mutagenesis on the synthesized PMA_RepCap vector (P190). The EaglII restriction enzyme also cuts NotI (IGEM standard) therefore EaglII has to be changed into a different recognition site. Primers were designed to change it into PvuII. The new QuikChange Lighting Site-Directed Mutagenesis Kit from Stratagene was used, therefore the mutagenesis was carried out as described in the QuikChange Manual. The PCR product was transformed into XL10-Gold cells, we used the IGEM standard protocol, except for the addition of ß-ME to the XL10-Gold cells.  :

    PCR-reaction:

    Volume / µl ingredientsrecommended /µl
    2,5 10X reaction buffer 2,5
    2,9 P190 template (~10 ng) 2,9 of 1:100 dilution
    0,51forward primer: O122 62,5 ng
    0,51 reverse primer: O123 62,5 ng
    0,5dNTP 250 µM each dNTP
    17,6H2O
    0,5QuikChange Lightning ennzyme(1.25)


    PCR program:

    Roundstemperature/ °CTime
    1 95 2 minutes
    18 9520 seconds
    18 60 10 seconds
    1868 2 minutes (4 kb * 30 sec)
    168 5 minutes


    The PCR product was digestet with Dpn I for 10 minutes at 37°C

    Plasmid was transformed into XL10-Gold cells.

    Sequencing results of Rep68, Rep78 and AAP BioBricks

    Investigator: Hanna
    Comment: pSB1C3_Rep78 (P160), pSB1C3_Rep68 (P165) and pSB1C3_AAP (P170) were sent for sequencing twice. No sequencing made sense. Paradoxically the Rep78 sequencing result fit to the p5 promoter BioBrick...
    By having a closer look to the BioBrick PCR we found out that the fragment sizes didn't fit to the expected sequences. In addition to that the test digestion also showed some discrepancies.
    Because we weren't able to find out in which steps of the BioBrick production things went wrong, we decided to discard all referring glycerol stocks and plasmids and to perform the Rep68, Rep78 and AAP BioBrick PCR again!!!

    Comment 13.08.2010 (Hanna): We wondered how the whole Rep stuff could be sequenced reverse AND forward without any forward sequencing primer... By checking the "sequencing book" we found out that always the wrong primers were used: the GOI primers can be used in order to sequence the Gene of interest = GOI (!) in pAAV_MCS and NOT the pSB1C3!!!!!!!!!


    Order oligos for CD-biobrick

    Investigator: Jessica and Kira
    ordered on aug 12th

    Freiburg10 oligos for CD-biobrick.jpg

    BioBrick assembly of pSB1C3_leftITR_CMV_mVenus

    Investigator: Stefan

    comment: In order to assemble our complete constructs mVenus has to be included into the pSB1C3_leftITR_CMV vector.
    Digestion:

    components: pSB1C3_leftITR_CMV P188(Vector) pGA14_mVenus P60 (Insert)
    DNA 6 8
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme1 1 (SpeI) 1 (Xba1)
    Enzyme2 1 (PstI)1 (PstI)
    H2O8 6
    Total volume 2020



    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 45 minutes


    Freiburg10 cloning of pSB1C3 leftITR CMV mVenus.png


    Expected sizes of constructs:

    • pSB1C3_leftITR_CMV: 2869 bp
    • pGA14_mVenus: 2896 bp, 756 bp

    The corresponding bands were cut out and Gel-Extraction was performed according to protocol.

    concentrations measured via NanoDrop:

    • pSB1C3_leftITR_CMV: 39,2 ng/µl
    • pGA14_mVenus: 8,2 ng/µl


    Ligation:

    T4 ligation was performed according to protocol.

    Volumes used:

    • vector: 1,67 ng/µl
    • insert: 6,33 ng/µl


    Transformation:

    bacterial strain used: XL1bB

    antibiotic used for plate: chlorampenicol

    Transformation was performed according to protocol.
    Plate was prepared and put in 37°C room.

    Opened a BioBrick box

    Investigator: Bea
    In order to "save" the plasmids/BioBricks which has been prepared and confirmed for submitting it to the parts registry a new box have been prepared. The box should contain 30 µL of the desired construct cloned into the pSB1C3 vector and the BBa number given from the headquarter.

    Freiburg10 BioBrick highlighting in Nomenclature list.JPG

    The first BioBricks which are in this box are:

    • pSB1C3_hGH (P136): Bba_K404002
    • pSB1C3_betaglobin (P133: Bba_K404001
    • pSB1C3_CMV (P145): Bba_K404003
    • pSB1C3_leftITR (P175): Bba_K404006
    • pSB1C3_rightITR (P172): Bba_K404005
    • pSB1C3_mGMK (P156): Bba_K404004

    In our nomenclature plasmid excel list the constructs which we aliquoted should be highlighted in blue and commented with the given Bba number. Additionally it should be taken in consideration that enough DNA should be in the "working" plasmid box. Therefore, if necessary, inoculate DYT medium for Mini-Preps.

    In this case ALL the glycerol stocks were used for incoluating 10 mL DYT medium containing 10 µL chloramphenicol respectively.

      To do: Mini-Preps of the over night cultures.


    88. labday 13.08.2010

    Seeding HT1080 for transduction at saturday, Seeding HEK293 for transfection

    Investigators: Kerstin, Adrian
    We seeded cells according to standard protocols.

    FACS of transduction from 11.8

    The YFP expression is still quite low. This was kind of anticipated, the success in this experiment is the fact, that the amount of living cells is at a very high level! (~90%). The conclusion: Detach the cells fast! Use PBS which is not at 37°C (the PBS was 25min in the water bath), transfer the cells fast on ice after detaching!

    The next steps are:

    • Is it possible, that freeze/thaw circles reduce the transduction efficiency?
    • Why is the YFP-expression that low? (~28-30%)

    Continuation:pSB1C3_SDM_SspI_bla14FM

    Investigator: Patrick
    Intention: receive enough plasmid for a sequencing and maybe futher working steps.
    Three clones were picked in the morning to inoculate DYT and perform a mini-prep in the evening and to send them for sequencing. The three clones were picked and prepped according to the standard protocol yielding the following DNA concentrations:

    • pSB1C3_SDM_SspI_bla14FM clone 1: 65,15 ng/µl , Sequencing labeled: PS3.1
    • pSB1C3_SDM_SspI_bla14FM clone 2: 22,3 ng/µl , Sequencing labeled: PS3.2
    • pSB1C3_SDM_SspI_bla14FM clone 3: 62,3 ng/µl , Sequencing labeled: PS3.3


    Used primer: seq_pSB1C3_VR2_rev (O51)
    Results: see http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal#Sequence_analysis_of_pSB1C3_SDM_SspI_BLA

    Mini-preps of pSB1C3_hgh,pSB1C3_beta globin,pSB1C3_left ITR,pSB1C3_right ITR,pSB1C3_mGMK,pSB1C3_CMV


    Comments:new mini-preps of the glycerol-stocks ofpSB1C3_hgh(=B103),pSB1C3_beta globin(=B100),pSB1C3_left ITR (=B143),pSB1C3_right ITR (=B140),pSB1C3_mGMK (=B125),pSB1C3_CMV  :


    componentsconcentration in ng/µl
    P191 (pSB1C3_right ITR) 100,74
    P192 (pSB1C3_beta-globin) 240,08
    P193 (pSB1C3_CMV) 333,89
    P194 (pSB1C3_hgh) 328,42
    P195 (pSB1C3_mGMK) 258,63
    P196 (pSB1C3_left ITR) 173,98

    Design and ordering of oligos for VP fusion targeting approaches

    Investigator: Hanna

    Comment: In order to target the EGFR receptor of our A431 cells different approaches have been figured out: Besides loop insertions, N-terminal fusion to the VP2 protein will be performed. In addition to that a VP1 insertion approach (after Grieger et al., 2007) was planned.
    Fusion and insertion molecules will be e.g. His-tag, Affibody, Darpin, GFP. These approaches can be combinated with the loop insertions.
    For this purpose the following oligos were ordered today:


    Freiburg10 N-terminalFusion1.jpg
    Freiburg10 N-terminalFusion2.jpg

    Mini-Prep and Test digestion of Rep40 and Rep52 of the cloning experiment ("Cloning of Rep40/52" 11.08.2010 Investigator: Anna)

    Investigator: Chris L., Achim

    Comments: There are small and big colonies on the plate. Maybe problems with the agar plate. Picked colonies of pSB1C3_Rep40 sample 1 and pSB1C3_Rep52 sample 1 are small colonies. Picked colonies of pSB1C3_Rep40 sample 2 + 3 and pSB1C3_Rep52 sample 2 + 3 are standard size colonies.


    Nanodrop

    • Rep 40 Sample 1: 156,26 ng/µl P197
    • Rep 40 Sample 2: 200,05 ng/µl P198
    • Rep 40 Sample 3: 144,06 ng/µl P199
    • Rep 52 Sample 1: 173,36 ng/µl P200
    • Rep 52 Sample 2: 160,75 ng/µl P201
    • Rep 52 Sample 3: 295,77 ng/µl P202



    Test digestion 800ng DNA

    components P145 /µl P146 /µl P145 /µl P146 /µlP145 /µl P146 /µl
    DNA 5,145,64,652,7
    BSA (10x) 222222
    Buffer 4 (10x) 222222
    Enzyme 1: XbaI 0,50,50,50,50,50,5
    Enzyme 2: SpeI 0,50,50,50,50,50,5
    H2O 9,9119,410,41012,3
    Total volume (e.g. 15,20,25,30 µl) 20 20 20 20 20 20


    Incubation time: 45 minutes, Incubation temperature: 37°


    0,5 g Agarose, 50ml x TAE, 3µl GELRED, at 115 Volt, running time: 45 minutes


    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 (Geneious) Expected size 2 (Geneious)
    Rep 40 1 20 µl 4 µl ____ bp 940 bp
    Rep 40 2 20 µl 4 µl ____ bp 940 bp
    Rep 40 3 20 µl 4 µl ____ bp 940 bp
    Rep 52 1 20 µl 4 µl ____ bp 1198 bp
    Rep 52 2 20 µl 4 µl ____ bp 1198 bp
    Rep 52 3 20 µl 4 µl ____ bp 1198 bp


    Marker Sample Rep40 1 /20µl Sample Rep40 2 /20µl Sample Rep40 3 /20µl Sample Rep52 1 /20µl Sample Rep52 2 /20µl Sample Rep52 3 /20µl
    Lane 1 2 3 4 5 6 7

    agarose gel: 1%, digestion: 45 minutes, 37°C


    Comments:Results look bad. Rep52 clone 3 looks good, send for sequencing.


    </p>
    

    Repetition of Rep 40 and Rep 52 ligation and transformation with the vector pSB1C3_CFP

    Investigator: Chris L.


    • Ligation of PCR products and vector:

    For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used.

    Sample-no.vector /µl insert /µl
    pSB1C3 + Rep40 1 4,92 3,08
    pSB1C3 + Rep52 2 6,47 1,53
    Total volume (e.g. 15,20,25,30 µl) 10 10


    • Transformation:

    The transformation was done following the standard protocol using XL1B cells.

    Strategy for VP2 N-terminal fusion and VP1 insertion of targeting moelcules

    Investigator: Hanna

    Comment: In order to target the EGFR receptor of tumor cells via Affibody or Darpin (antibody fragment is also possible!!!), or in order to expose e.g. His-Tags on the virus surface, two approaches have been figured out.

    Freiburg10 N-terminalFusion Strategy.JPG
    Freiburg10 N-terminalFusion Strategy2.JPG


    <p style="font-size:15px; background-color:#66bbff;">pSB1C3_Rep52 (P202) sent for sequencing

    Investigator: Chris L.

    Primer used: Reverse Primer VR2

    Repetition: Biobrick production of Rep 68, 78 and AAP

    Investigator: Stefan


    Aim of the experiment: We want to produce biobricks from Rep 68, Rep 78 and the AAP.

    • Plasmids used as template:

    Rep_68_ex (p119): c = 470,6 ng/µl
    Rep_78_(p122): c = 566,8 ng/µl
    pAAV-RC containing AAP ORF (p50): c = 378,5 ng/µl


    • Primer used:

    For Rep_68_ex: Praefix_68_78_ex & Suffix_40_68_ex
    For Rep_78_ex: Praefix_68_78_ex & Suffix_52_78_ex
    For AAP_ex: Praefix_AAP_ex & Suffix_AAP_ex


    • PCR:

    (was performed following the standard protocol)

    Ingredients Volume / µl Rep68Rep78 AAP
    5X Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: 2,5
    reverse primer: 2,5
    DNA Template***2,5 µl 2 µl3 µl
    DMSO (2%) - - 1 µl
    Phusion Polymerase0,5
    H2O*** 31 µl 31,5 µl29,5 µl
    Total volume50


    PCR program:

    PCR Programtemperature/ °CTime Rep68Rep78AAP
    198 1min
    298 15s
    3*** 25s63°C62°C64°C
    4 (step 2-4 8x) 72***24s 27s 10s
    5 98 15s
    6***25s68°C64°C68°C
    772***24s27s10s
    8 (step 6-8 17x)725min
    Hold4


    Used agarose gel: 0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:45 minutes

    Freiburg10 68 78 AAP.jpg


    Gel extraction


    Gel extraction was perfomed according to protocol.

    • Digestion of PCR products and vector:


    components PCR products /µl vector /µl
    DNA 29 11
    BSA (10x) 4 2
    Buffer 4 (10x)4 2
    Enzyme XbaI 1 1
    Enzyme SpeI 1 1
    H2O1 3
    Total volume (e.g. 15,20,25,30 µl) 4020


    Comments: Digestion was done with XbaI and SpeI, it has to be checked if the inserts are cloned into the vector in the right orientation.


    • Purification of Rep68, 78 and AAP:

    For the purification 200 µl of buffer PBI was used.

    c(Rep68)= 14,3 ng/µl
    c(Rep78)= 12,0 ng/µl
    c(AAP)= 25,1 ng/µl


    • Gelextraction of pSB1C3_RFC25_CFP:

    0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:50 minutes
    4µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)



    Freiburg10 pSB1C3 CFP 130810.jpg




    c(pSB1C3)= 6,9 ng/µl



    • T4 ligation of PCR products and vector:

    For the Ligation 1µl buffer (10x) and 1µl T4 ligase were used.

    vector /µl insert /µl
    pSB1C3 + Rep68 3,78 4,24
    pSB1C32 + Rep78 3,13 4,87
    pSB1C3 + AAP 6,42 1,58


    • Transformation:

    The transformation was done following the standard protocol using XL1b cells.

    89. labday 14.08.2010

    Seeding HEK293 for transfection at monday 16.8

    Investigator: Kerstin, Adrian
    We want to knoe which confluence of HEK293 cells is optimal for AAV production.

    The plan
    We seed different amounts of cells in 10 cm2, and check the highest titer.

    Plate amount of cells
    1100 000
    2200 000
    3400 000
    4500 000
    5800 000
    61 000 000
    71 200 000
    81 500 000
    91 750 000
    101 750 000

    Transduction with mVenus loaded viral particles

    Investigator: Kerstin, Adrian

    the transduction was performed with following viral stocks:


    Plate pAAV_RC/µgpHelper/µgGOI/µg
    13,3 3,3 no GOI
    26,6 3,3 no GOI
    33,3 3,3 3,3 TK_GMK clone1
    43,3 3,3 3,3 TK_GMK clone1
    53,3 3,3 3,3 TK_GMK clone2
    63,3 3,3 3,3 YFP
    73,3 3,3 10 YFP
    83,3 3,3 20 YFP
    93,3 3,3 40 YFP
    103,3 3,3 60 YFP

    Platte 1:

    1 2 3
    A control, no cells 300µl virus 1 300µl virus 2
    B control, no virus 600µl virus 1 600µl virus 2


    Platte 2:

    1 2 3
    A control, no cells 300µl virus 3 300µl virus 4
    B control, no virus 600µl virus 3 600µl virus 4


    Platte 3:

    1 2 3
    A control, no cells 300µl virus 5 300µl virus 6
    B control, no virus 600µl virus 5 600µl virus 6


    Platte 4:

    1 2 3
    A control, no cells 300µl virus 7 300µl virus 8
    B control, no virus 600µl virus 7 600µl virus 8


    Platte 5:

    1 2 3
    A control, no cells 300µl virus 9 300µl virus 10
    B control, no virus 600µl virus 9 600µl virus 10


    Platte 6:

    1 2 3
    A control, no cells 300µl virus 3 300µl virus 4
    B control, no virus 600µl virus 3 600µl virus 4


    Platte 7:

    1 2 3
    A control, no cells 300µl virus 3 300µl virus 4
    B control, no virus 500µl virus 3 500µl virus 4


    Platte 8:

    1 2 3
    A control, no cells 300µl virus 5 300µl virus 5
    B control, no virus 600µl virus 5 600µl virus 5


    The test for functionality for TK GMK was done with the Prodrug Cymeven ganciclovir 500mg from Roche. The final concentration was 0,05 mM in the wells. After two days pictures were taken.


    Sequence analysis of pSB1C3_SDM_SspI_BLA

    Investigator: Patrick, Bea

    Comments: Sequence analysis of three clones revealed that all sequences contain some mutations in the regions where the primer annealed. This was due to the extreme primer lentgh. Primers that long should be ordered as HPLC purified in order to avoid mutations in the primer sequence.

    1. Clone 1: Mutation in the SalI site of the loop insertion site in the Viral Brick MCS lead to the removal of the recognition site. CANNOT be used for the loop insertions!!!!
    2. Clone 2: Mutation in the region of the primer upstream of the loop insertion sites SalI and PvuII.
    3. Clone 3: Mutation in the region of the primer downstream of the loop insertion sites SalI and PvuII.



    Next steps: Two additional clones will be picked and sent for sequencing (clones 4 and 5). In order to obtain a construct without any mutations, clone 2 and clone 3 couldbe cloned together. Each clone will be digested with SalI and BamHI. Clone 2 will be the "insert", clone 3 is going to be the "vector". Therefore the fragments will (hopefully) not contain anymore mutations in the relevant sequence.

    Mini Preps of PMA_RepCap vector and pSB1C3_left ITR_CMV_betaglobin_mVenus


    Investigator: Chris L., Bea

    Comments:new mini-preps of the glycerol-stocks of pSB1C3_leftITR_CMV_betaglobin_mVenus clone 1 (=B179), pSB1C3_leftITR_CMV_betaglobin_mVenus clone 2 (=B180), pSB1C3_leftITR_CMV_betaglobin_mVenus clone 3 (=B181), pMA_RepCap Vector_SDM_InsPvuII clone 1, (=B182), pMA_RepCap Vector_SDM_InsPvuII clone 2 (=B183) and pMA_RepCap Vector_SDM_InsPvuII clone 3 (=B184)


    Comment: The plamsid numbers in the picture (P179-P184) correspond to the glycerol stocks B179-B184. The corresponding plasmid numbers are: P208-P213.
    Nanodrop

    • pSB1C3_leftITR_CMV_betaglobin_mVenus clone 1: 256,64 ng/µl B179
    • pSB1C3_leftITR_CMV_betaglobin_mVenus clone 2: 274,86 ng/µl B180
    • pSB1C3_leftITR_CMV_betaglobin_mVenus clone 3: 217,20 ng/µl B181
    • pMA_RepCap Vector_SDM_InsPvuII clone 1: 253,71 ng/µl B182
    • pMA_RepCap Vector_SDM_InsPvuII clone 2: 195,64 ng/µl B183
    • pMA_RepCap Vector_SDM_InsPvuII clone 3: 144,61 ng/µl B184



    Test digestion 800ng DNA

    components B179 /µl B180 /µl B181 /µl B182 /µlB183 /µl B184 /µlpMA_RepCap Vector /µl
    DNA 3,12,93,73,145,5 2,4
    BSA (10x) 2222222
    Buffer 4 (10x) 2222222
    Enzyme 1: NgoMIV 0,50,50,5000 0
    Enzyme 2: EcoRI 0,50,50,50000
    Enzyme 2: PvuII 000 0,50,50,50,5
    H2O 11,912,111,312,411,510 13,1
    Total volume (e.g. 15,20,25,30 µl) 20 20 20 20 20 20 20


    Incubation time: 45 minutes, Incubation temperature: 37°


    0,5 g Agarose, 50ml x TAE, 3µl GELRED, at 115 Volt, running time: 45 minutes


    Results of test digestion

    Freiburg10 Test digestion of pSB1C3 Bba fourparts SDM pMA RC insert 14 08.jpg


    Results: Two (three) different approaches can be seen on the agarose gel.

    1. P178-P181 (blue): The plasmid pSB1C3_leftITR_CMV_betaglobin_mVenus was digested with NgoMIV and EcoRI. Expected bands were: 2873bp and 1343 . As it can be seen in the picture, the two fragments ran too high (~300bp). As it is almost normal for the pSB1C3 plasmid, we should figure out why this is always the problem for this vector.
    2. P182-P183 (red): The plasmid pMA_RepCap_SDM_insPvuII was digested with PvuII as well as the control vetor pMA_RepCap where no site-directed mutagenesis was performed in order to see the difference. As it can be seen in the picture above, the expected band sizes of 263bp,1384bp and 2133bp correspond to the bands detected in the gel. The control vector instead reveals only two bands detectable. Therefore site-directed mutagenesis was successfully performed.
    3. P177 (green): pSB1C3_hTERT. For further details see extra part below (Test digestion of pSB1C3_hTERT.

    Mini-Prep of pSB1C3_leftITR_CMV_mVenus

    Investigator: Stefan

    Glycerol stocks were prepared:

  • B177 = pSB1C3_leftITR_CMV_mVenus clone 1
  • B178 = pSB1C3_leftITR_CMV_mVenus clone 2 Mini-Prep following the standard protokol
  • P206 = pSB1C3_leftITR_CMV_mVenus clone 1 = 277,9 ng/µl
  • P207 = pSB1C3_leftITR_CMV_mVenus clone 2 = 260,3 ng/µl


    Test digestion: or test digestion, pSB1C3_leftITR_CMV (P188) was digested as well.
    Ingredients clone 1/µlclone 2/µl P188/µl
    DNA: 3,5 3,5 4
    BSA (10x): 2 2 2
    Buffer 4: 2 2 2
    Enzyme 1 EcoRI: 0,5 0,5 0,5
    Enzyme 2 AgeI0,5 0,50,5
    H2O 6,5 6,56


      All samples were loaded on a 1% agarose gel:
    • GeneRuler Ladder Mix (7µl)
    • P206 = pSB1C3_leftITR_CMV_mVenus clone 1
    • P207 = pSB1C3_leftITR_CMV_mVenus clone 2
    • P188 = pSB1C3_leftITR_CMV


    Freiburg10 test digestion leftITR CMV mVenus 140810.jpg


    comment: Through digestion, fragments would have similar sizes. Therefore, a new digestion will be performed tomorrow.

    Test digestion of pSB1C3_pHTERT

    Investigator: Bea

    Comments: pSB1C3_hTERT was sent for sequencing and revealed that the PCR and the inserting of the PCR product : promoter for hTERT was sucessfully performed. But the sequencing results showed a new AgeI restriciton site in the plasmid backbone. We could not see if the new recognition site was due to the bad sequence read or that there is really a new AgeI site. Therefore, a test digestion will be perfomed with PstI and AgeI in order to figure our wheter AgeI is present in the backbone or not.


    Sequence analysis of pSB1C3_hTERT:



    First the sequenced plasmid P177 was digested:

      P177/µL
    DNA 3
    BSA (10x) 1,5
    Buffer 4 (10x) 1,5
    Enzyme PstI 0,5
    Enzyme AgeI 0,5
    H2O 8
    Total volume 15


    • Incubation time: 45minutes
    • Incubation tempereature: 37°C

    After the incubation,the samples were loaded on the 1% agarose gel, and run at 110 V for 45 minutes.

    Result of the test digestion:


    Results: As it can be seen in the sequencing results of pSB1C3_hTERT there could have been an additional AgeI site in the vector. If AgeI would have been in the vector, two fragments have been expected by digetsing the plasmid with AgeI and PstI. As it can be seen in the agarose gel, lane 11 (labeled with the green P177) only reveals one lane instead of two. Therefore it can be assumed that NO AgeI site is in the pSB1C3 backbone.

    Picking clones ...

    Investigator: Patrick
    15 Clones were picked saturday night so that a mini-prep could be performed on sunday.

    90. labday 15.08.2010

    Sequence analysis of pSB1C3_Rep52

    Investigator: Bea

    Comments: Clone 3 was sent for sequencing because this clone revealed the right band sizes of the test digestion performed friday. For more details see: Test digestion


    Results:

    Sequence assembly showed that the suffix of the Rep protein 52 is ok and that PCR worked well.

    There can be seen that a silent mutation from AGG to AGA was inserted, but it will have no effect on the protein because codon usage in H. sapiens is nearly the same for both codons.

    The prefix could not be analysed because of bad sequence read quality. For further verification, another sequencing with the forward primer has to be performed.
    Next steps: Send plasmid for another sequencing round with forward primer in order to analyse the prefix.

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    Mini-Prep of pSB1C3_Rep68, pSB1C3_Rep78, pSB1C3_RepAAP, pSB1C3_Rep40, pSB1C3_Rep52

    Investigator: Stefan

      Glycerol stocks were prepared:
    • B185 = pSB1C3_Rep68 clone 1
    • B186 = pSB1C3_Rep68 clone 2
    • B187 = pSB1C3_Rep68 clone 3

    • B188 = pSB1C3_Rep78 clone 1
    • B189 = pSB1C3_Rep78 clone 2
    • B190 = pSB1C3_Rep78 clone 3

    • B191 = pSB1C3_AAP clone 1
    • B192 = pSB1C3_AAP clone 2
    • B193 = pSB1C3_AAP clone 3

    • B194 = pSB1C3_Rep40 clone 1
    • B195 = pSB1C3_Rep40 clone 2

    • B196 = pSB1C3_Rep52 clone 1
    • B197 = pSB1C3_Rep52 clone 2

    • B198 = pSB1C3_SDM_SspI_Bla14FM clone 4
    • B199 = pSB1C3_SDM_SspI_Bla14FM clone 5



      Mini-Prep following the standard protokol
    • P208 = pSB1C3_Rep68 clone 1 c=230,0
    • P209 = pSB1C3_Rep68 clone 2 c=160,5
    • P210 = pSB1C3_Rep68 clone 3 c=128,6

    • P211 = pSB1C3_Rep78 clone 1 c=150,8
    • P212 = pSB1C3_Rep78 clone 2 c=108,6
    • P213 = pSB1C3_Rep78 clone 3 c=131,3

    • P214 = pSB1C3_AAP clone 1 c=142,1
    • P215 = pSB1C3_AAP clone 2 c=148,3
    • P216 = pSB1C3_AAP clone 3 c=163,0

    • P217 = pSB1C3_Rep40 clone 1 c=164,5
    • P218 = pSB1C3_Rep40 clone 2 c=<145,1br />
    • P219 = pSB1C3_Rep52 clone 1 c=156,0
    • P220 = pSB1C3_Rep52 clone 2 c=164,0

    • P221 = pSB1C3_SDM_SspI_Bla14FM clone 4 c=220,4
    • P222 = pSB1C3_SDM_SspI_Bla14FM clone 5 c=252,2




    Test digestion:
    No test digestion for pSB1C3_SDM_SspI_Bla14FM clone 4 and 5.
    For test digestion, pSB1C3_leftITR_CMV_mVenus (P206-207) and pSB1C3_leftITR_CMV (P188) was digested as well.
    Samples P208-220 were digested with XbaI and SpeI.
    Samples P207, P207 and P188 were digested with XbaI and NgoMIV.


    Gel used:
    100 ml TAE (1x), 1 g agarose, 6 µl GELRED
    115 V, 50 minutes

    Ingredients P208/µlP209/µl P210/µl P211/µlP212/µl P213/µlP214/µl P215/µlP216/µl P217/µlP218/µl P219/µlP220/µl P206/µlP207/µl P188/µl
    DNA: 46 7 6 9 7 6 6 66 6 6 6 3,5 3,5 4
    BSA (10x): 2 2 22 2 22 2 22 2 22 2 22
    Buffer 4: 2 2 22 2 22 2 22 2 22 2 22
    Enzyme 1 0,5 0,5 0,50,5 0,5 0,50,5 0,5 0,50,5 0,5 0,50,5 0,5 0,50,5
    Enzyme 2 0,5 0,5 0,50,5 0,5 0,50,5 0,5 0,50,5 0,5 0,50,5 0,5 0,50,5
    H2O 11 98 9 68 9 99 9 99 9 11,511,5 11



    Freiburg10 Rep40-78 mVenus.jpg


    comment: Rep- and AAP approaches have to be repeated. We did not realize that the vector can religate! Plasmids and glycerol stocks were thrown away.

    Inoculation of CD colony

    Investigator: Kira

    One colony was picked from agar plate and dispersed into 10 ml DYT+ Amp and incubated over night @ 37 C.

    91. labday 16.08.2010

    Minipreps of P119, P122, P204, P205

    Investigator: Achim

    Cloning of pSB1C3_SspIdel_BLA clone 2 & 3 to get rid of mutations

    Investigator: Achim

    • Sequencing showed that clone 2 had a mutation in the vector, clone 3 had one in the BLA sequence. Therefore, both were cut with BamHI and SalI, then the BLA fragment of clone 2 and the vector fragment of clone 3 were ligated. The final construct shouldn't contain any more mutations.

    Freiburg10 16082010achim.jpg

    Nice picture, but where's the protocol? Results? (Hanna) ;)

    New working solutions were prepared

    Investigator: Patrick

    • DMEM
    • Amp
    • H2O millipore
    • DYT

    Further pSB1C3_SDM_SspI_Bla14FM sequencing

    Investigator: Patrick
    Two additional Clones were picked (clones 4 & 5) and sent for sequencing, labeled: iGEM4.1 and iGEM4.2.
    Used Primer: seq_pSB1C3_VR2_rev (O51)

    Mini prep of pKS-CD

    Investigator: Kira

    c(pKS-CD) = 291, 39 ng/µl

    Yielded DNA was used for site directed mutagenesis


    Fluorescence microscopy of TKGMK loaded particles treated cells

    The fluorescence was quite low (~ 5%)in nearly every well! The 40µg YFP wells were the only exception.

    Remember: This was just a qualitative check of our constructs! The scale bar is missing but its not that dramatic. As you can see, our construct works! It is possible to kill tumor cells with our vector.

    SDM of PstI in pKS-CD

    Investigator: Jessica, Kira

    Motivation: the desired gen contains 2 iGEM restrictions sites, thus in oder to use it for further cloning, these site have to be deleted. The first SDM was performed on PstI restriction site.

    • P223 pKS_CD
    • O154 CD_PstI FP
    • O155 CD_PstI RP
    component Volume/µl
    DNA: (1:20) 0,5
    10x reaction buffer: 2,5
    primer forward PstI (1:10) 0,56
    primer reverse PstI (1:10) 0,56
    dNTP 0,5
    DMSO 0,5
    H2O 14,38
    PfuTurbo DNA Polymerase 0,5


    PCR

    segment cycles temperature time
    1 1 95 2min
    2 20 95 30sec
    55 1min
    68 4 min30 sec
    3 4 hold


    sample was digested with DpnI for 1h at 37°C

    Transformation was performed according to the standard protocol.

    Biobrick production of pSB1C3_lITR_CMV_beta-globin_mVENUS_hgh_rITR and pSB1C3_lITR_CMV_mVENUS_hgh_rITR

    Investigator: Anna, Anissa

    Comments: hgh_rITR was cloned one time into pSB1C3_lITR_CMV_beta-globin_mVENUS and one time into pSB1C3_lITR_CMV_mVENUS to receive a control for the improtance of beta-globin

    • Digestion:
    components volume of pBS1C3_hgh_rITR INSERT = p186 /µl volume of pBS1C3_lITR_CMV_beta-globin_mVENUS VECTOR = p208 /µlvolume of pBS1C3_lITR_CMV_mVENUS VECTOR = p207 /µl
    DNA 196 3,84
    BSA (10x) 3 31,5
    Buffer 4 (10x)3 3 1,5
    SpeI° /XbaI*1*
    PstI1 11
    H2O3 6 6,16
    Total volume3020 15


    The samples were digested for 2 h at 37°C

    • Gel: samples were loaded on a 1% agarose-gel. running time: 45 minutes

    Freiburg10 psb1c3 lITR CMV ß-globin mVenus hgh rITR und psb1c3 lITR CMV mVenus hgh rITR.png

    • gelextraction was performed according with the standard-protocol


    • Ligation:
    components concentration/ ng/µl
    p186 8,97
    p207 25,57
    p20817,73


    components amount for ligation /µl
    p186 (for p207)4,2
    p207 3,8
    p186 (for p208)4,7
    p2083,3
    T4 ligation buffer1
    T4 Ligase1
    • Transformation: was performed according with the standard-protocol

    Preparation for Midi-Preps of B24 and B34

    Investigator: Anna

    • 40 ml DYT was prepared with 40 µl Ampicillin and inoculated with B24 and B34, both were incubated over night in 37°C room.

    What is B24 and B34? Can you please be more precise - I also want to understand what's happening also when I am not able to check up in the excel sheets ;) (Hanna)

    Cloning of pAAV_RC (P50) with pMA_RC_insert (P190)

    Investigator: Chris L.

    • Vector: name: pAAV_RC P50
    • Insert: name: pMA_RC_insert P190
    • buffer used: 3
    • Restriction-enzymes used:
      • BstEII (no. Lab: 17)
      • SwaI (no.Lab: 135)
    • DNA concentration (P50): 378,5 ng/µl
    • DNA concentration (P190): 339,5 ng/µl


    Digestion:

    components volume of pMA_RC_insert /µl volume of pAAV_RC /µl
    DNA 4,4 2,6
    BSA (10x) 2 2
    Buffer 3 (10x)2 2
    SwaI (no.Lab:135)0,5 0,5
    BstEII (no.Lab:17)0,5 0,5
    H2O10,6 12,4
    Total volume 2020


    Comments: Incubation at 25° after addition of SwaI for 1,5 hours. Then incubation at 60° after addition of BstEII for 1,5 hours.


    Gel extraction:
    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

    Expected sizes of constructs:

    • pAAV_RC 6839 bp and 493 bp
    • pMA_RC_Insert 3292 bp and 493 bp


    Marker Sample P190, 20µl Sample P 50, 20µl
    Lane 3 5 7


    Results:

    Bands are really weak, tomorrow I`ll take more DNA in sample.
    Next steps: Tomorrow repetition of experiment


    Repetition: Biobrick production of Rep40, Rep68, Rep78 and AAP

    Investigator: Stefan


    Aim of the experiment: We want to produce biobricks from Rep40, Rep68, Rep78 and AAP.

    • Plasmids used as template:

    Rep_68_ex (p119): c = 470,6 ng/µl
    Rep_78_(p122): c = 566,8 ng/µl
    pAAV-RC containing AAP ORF (p50): c = 378,5 ng/µl


    • Primer used:

    For Rep_68_ex: Praefix_68_78_ex & Suffix_40_68_ex
    For Rep_78_ex: Praefix_68_78_ex & Suffix_52_78_ex
    For AAP_ex: Praefix_AAP_ex & Suffix_AAP_ex

    • PCR:

    (was performed following the standard protocol)

    Ingredients Volume / µl Rep68Rep78 AAPRep40
    5X Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: 2,5
    reverse primer: 2,5
    DNA Template***2,5 µl 2 µl3 µl1 µl
    DMSO (2%) - - 1 µl -
    Phusion Polymerase0,5
    H2O*** 31 µl 31,5 µl29,5 µl 31,5 µl
    Total volume50


    PCR program:

    PCR Programtemperature/ °CTime Rep68Rep78AAP Rep40
    198 1min
    298 15s
    3*** 25s63°C62°C64°C63°C
    4 (step 2-4 8x) 72***24s 27s 10s 15s
    5 98 15s
    6***25s68°C64°C68°C 68°C
    7 (step 5-7 17x)72***24s27s10s 15s
    8725min
    Hold4°C


    Used agarose gel: 0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time:45 minutes

    Freiburg10 Rep40 68 78 AAP.png

    Comments: I forgot to dilute Rep68. Therefore, a new PCR will have to be performed tonight.



    Gel extraction


    Gel extraction was perfomed according to protocol.


    • Digestion of PCR products and vector:


    components PCR products /µl vector /µl
    DNA 29 11
    BSA (10x) 4 2
    Buffer 4 (10x)4 2
    Enzyme XbaI 1 1
    Enzyme SpeI 1 1
    H2O1 3
    Total volume (e.g. 15,20,25,30 µl) 4020


    Comments: Digestion was done with XbaI and SpeI, it has to be checked if the inserts are cloned into the vector in the right orientation.


    • Purification of Rep40, 78 and AAP:

    For the purification 200 µl of buffer PBI was used.

    c(Rep40)= 6,5 ng/µl
    c(Rep78)= 14,3 ng/µl
    c(AAP)= 39,6 ng/µl


    • Gelextraction of pSB1C3_RFC25_CFP:

    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time:50 minutes
    4µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)



    Freiburg10 pSB1C3 2x.jpg




    c(pSB1C3)= 19,5 ng/µl



    • T4 ligation of PCR products and vector:

    For the Ligation 1µl buffer (10x) and 1µl T4 ligase were used.

    vector /µl insert /µl
    pSB1C3 + Rep40 1,57 6,43
    pSB1C32 + Rep78 1,71 6,29
    pSB1C3 + AAP 5,56 2,44


    • Transformation:

    The transformation was done following the standard protocol using XL1b cells.

    Design and Ordering of Oligos

    Investigator: Hanna

    SV40 terminator

    Comment: The CMV promoter is often used in combination with a SV40 terminator. Because we want to use this promoter in the context of the VP2 fusion and VP1 insertion approaches, oligos were designed in order to produce a SV40 terminator BioBrick (from the pEGFP-C1 plasmid).
    Freiburg10 SV40Terminator.jpg

    pEGFP_backbone

    Comment: In order to perform the VP2 N-terminal fusion and VP1 insertion approaches, we decided to use pEGFP-C1 as expression plasmid. This plasmid contains an EGFP under the control of the CMV promoter and the SV40 terminator. In order to exchange the EGFP with our targeting constructs a PCR of the CMV_backbone_SV40Terminator should be performed. The referring primers were designed with EcoRI and PstI restriction sites. One should keep in mind that this expression plasmid mustn't digested with NgoMIV and it contains Kanamycin-resistance!
    Freiburg10 pEGFP-C1 backbone.jpg

    92. labday 17.08.2010

    Overview of the state-of the art plasmid preparation

    Investigator: Bea

    Comments: In order to obtain a overview (more or less plausible ;-)) I sorted out how the final plasmid need to be constructed. In the picture you can see the most important plasmids which we will need for the retargeting approaches starting tomorrow.



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    Further pSB1C3_SDM_SspI_Bla14FM sequencing

    Investigators: Bea, Patrick

    Sequencing results: there is no BLA in clone 4. Clone 5 showed a flawless BLA so it will be used for the next site directed mutagenesis.

    pSB1C3_SDM_SspI_Bla14FM clone 5 SDM (P222)

    Investigator: Patrick
    Intention: remove PvuII restriction site

    PCR program:

    • 95°C 2 min (1x)
    • 95°C 20s, 60°C 10 s, 68°C 30 s (30s/kb) (18x)
    • 68°C 5 minutes
    Ingredients Volume
    10x reaction buffer 2,5
    DNA template ( about 10 ng) 1 µl diluted P222
    forward primer: 0,58 µl O108 (pSB1C3 PvuII rev)
    reverse primer: 0,59 µl O109 (pSB1C3 PvuII for)
    DMSO (2%) 0,5
    dNTP Mix from the kit 0,5 µl
    QuickSolution Reagent0,75 µl
    Quickchange Lightning Enzyme (1.25U)0,5 µl
    H2O 18,08 µl
    Total volume25 µl


    Further procedure including the transformation was performed according to the standard protocol.

    Sent for sequencing

    • pMA_RC (P222) sent for sequencing with O41 (Cap 4000 for) to check if the EaglII restiction site was successfully changed to PvuII.
    • pAAV_RC (P158) sent for sequencing with GATC_std_SK, Rep_1250 for (O35), GATC_std_UP-2 to check if all deleted restriction sites are present (PstI, BamHI, SalI).

    Investigators: Bea, Patrick

    Cloning of pMA_RC-Insertparts in pAAV_RC 1.2 SDM SalI

    Investigator: Jessica
    P211 will be cloned in the P158 vector that contains 4 mutation (BamHI, SalI, PstI 320 + 4073)
    Digestion:

    components pMA_RC_Insertparts pAAV_RC 1.2 SDM SalI
    DNA 5 4
    BSA (10x) 1 1
    Buffer 3 (10x)1 1
    Enzyme1 BstEII 0,5 0,5
    Enzyme2 SwaI0,5 0,5
    H2O2 3
    Total volume1010


    digestion was splitted in two parts:
    • 1. digestion with SwaI at 25°C for 1,5h
    • 2. digestion with BstEII at 60°C for 1,5h

    Preparation of gel:
    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at Volt, running time: minutes

    Expected sizes of constructs:

    • pMA_RC-Insertparts: 493bp, 3287bp
    • pAAV_RC 1.2 SDM SalI: 6839bp, 478bp

    The corresponding bands were cut out and Gel-Extraction was performed according to protocol.
    Freiburg1 cloning of pMA RC-Insertparts in pAAV RC 1.2 SDM SalI.jpg

    concentrations measured via NanoDrop:

    • pMA_RC-Insertparts: 5,9 ng/µl
    • pAAV_RC 1.2 SDM SalI:8,6 ng/µl


    Ligation
    Volume insert: 1,95 µl
    Volume vector: 6,08 µl

    Trafo was prepared with BL21

    Repetition: Biobrick production of Rep 68

    Investigator: Christian L. , Stefan


    Aim of the experiment: We want to produce biobricks from Rep 68.

    • Plasmid used as template:

    Rep_68_ex (p224): c = 350,29 ng/µl


    • Primer used:

    For Rep_68_ex: Praefix_68_78_ex & Suffix_40_68_ex

    • PCR:

    (was performed following the standard protocol)

    Ingredients Volume Rep68 / µl
    5x Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: 2,5
    reverse primer: 2,5
    DNA Template3
    DMSO (2%) -
    Phusion Polymerase0,5
    H2O 30,5
    Total volume50


    PCR program:

    PCR Programtemperature/ °CTime
    198 1min
    298 15s
    363 25s
    4 (step 2-4 8x) 7224s
    5 98 15s
    66825s
    7 (step 5-7 17x)7224s
    8725min
    Hold4°C


    Used agarose gel: 0,5 g Agarose,50 ml TBE (1%), 3 µl GELRED , at 115 Volt, running time:45 minutes

    Freiburg10 Rep68.jpg


    Gel extraction was perfomed according to protocol.

    comment: Elution was put in 4°C room. Work will be continued tomorrow.

    Transduction of HT1080 cells

    Transduction plan

    Investigator: Adrian, Kerstin

  • 1. plate 50.000 cells, YFP
    A control no cells 300 µl virus (40 µg YFP) 300 µl virus (20 µg YFP)
    B control no virus 500µl P38 (pAAV2_mVenus) 1000µl P38 (pAAV2_mVenus)
  • 2. plate 50.000 cells, YFP
    A control no cells 300 µl virus (40 µg YFP) 300 µl virus (20 µg YFP)
    B control no virus 500µl P38 (pAAV2_mVenus) 1000µl P38 (pAAV2_mVenus)
  • 3. plate 100.000 cells, YFP
    A control no cells 300 µl virus (40 µg YFP) 300 µl virus (20 µg YFP)
    B control no virus 500µl P38 (pAAV2_mVenus) 1000µl P38 (pAAV2_mVenus)
  • 4. plate 100.000 cells, YFP
    A control no cells 300 µl virus (40 µg YFP) 300 µl virus (20 µg YFP)
    B control no virus 500µl P38 (pAAV2_mVenus) 1000µl P38 (pAAV2_mVenus)
  • 5. plate 100.000 cells TKGMK
    A control no cells 150 µl virus (TKGMK clone 1) 180 µl virus (TKGMK clone 1)
    B control no virus 200 µl (TKGMK clone2) 200µl (no GOI)

    Midi-Prep of pAAV_igEM_mVenus_YFP and dsAAV_CMV_EGFP

    Investigators: Chris, Anna

    Comment: Midi-Preps of dsAAV_CMV_EGFP (p31, B24) and pAAV_iGEM_mVenus_YFP (p39, B34)

    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    plasmid-no. p31p39
    concentration (ng/µl)539,05 162,37


    BioBrick assembly for pSB1C3_lITR_CMV_beta-globin_YFP_rITR

    Investigator: Achim

    • This construct without the hGH polyadenylation sequence will be used to determine wether/how much hGH affects translation of the virus gene.

    Vector:

    • pSB1C3_lITR_CMV_ß_YFP (P209): c=274,86 ng/µl

    Insert:

    • pSB1C3_rITR (P172/P191): c=73,3 ng/µl


    components VectorInsert
    DNA 3,6 20,5
    BSA (10x) 3 3
    Buffer 4 (10x)3 3
    Enzyme 1 1(SpeI) 1(XbaI)
    Enzyme 2 1(PstI) 1 (PstI)
    H2O18,4 1,5
    Total volume 30 30


    • 1% Agarose gel
    • 3 µl Gelred
    • 7 µl DNA-Ladder-Mix
    • 115 Volt, running time: 30 minutes

    The Insert containing the right ITR was cut out after 30 minutes, the Vector was cut out after 45 minutes

    Freiburg10 Achim 17 8 10.jpg



















    Sample/µl Expected size/bp
    Vector 4100
    Insert 150


    • weight of insert gel extract: 170 mg
    • weight of vector gel extract: 200 mg


    Nanodrop

    • insert : 11,4 ng/µl
    • vector : 26,7 ng/µl


    Ligation

    • vector: 6,36 µl
    • insert: 1,64 µl

    Transformation

    BioBrick assembly for pSB1C3_lITR_pTERT

    Investigator: Anissa

    • Construct with the pTERT tumor specific promoter

    Vector:

    • pSB1C3_lITR (P196): 174 ng/µl

    Insert:

    • pSB1C3_phTERT (P177): c=312 ng/µl


    components VectorInsert
    DNA 5,75 6,41
    BSA (10x) 1,5 1,5
    Buffer 4 (10x)1,5 1,5
    Enzyme 1 1(SpeI) 1(XbaI)
    Enzyme 2 1(PstI) 1 (PstI)
    H2O4,25 3,59
    Total volume 15 15


    • 1% Agarose gel
    • 3 µl Gelred
    • 7 µl DNA-Ladder-Mix
    • 115 Volt, running time: 45 minutes


    Freiburg10 Achim anissa 17 8.jpg



















    Sample/µl Expected size/bp
    Vector 2200
    Insert 500


    • weight of insert gel extract: 350 mg
    • weight of vector gel extract: 250 mg


    Nanodrop

    • insert : 17,7 ng/µl
    • vector : 27,6 ng/µl


    Ligation

    • vector: 5,57 µl
    • insert: 2,43 µl

    Transformation

    Picking clones of pSB1C3_lITR_CMV_(betaglobin)_mVenus_hGH_rITR, EGFP_C1 and pMA_T_affibody

    Investigator: Anna

    Plasmid Resistance Bacterial strain
    pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR° ChloramphenicolXL1 blue
    pSB1C3_lITR_CMV_mVEnus_hGH_rITR° ChloramphenicolXL1 blue
    EGFP_C1Kanamycin
    pMA_T_affibody Ampicillin

    °: 3 clones each

    Clones were inoculated in 10 mL DYT medium containing 10 µL of antibiotics and were put in 37°C room on shaker.

    Trafo evaluation of SDM PstI in CD and inoculation

    Investigator: Kira

    The plate contained lots of colonies and 2 of them were picked and inoculated into DYT+Amp



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